CA2746001C - Treatment of sirtuin 1 (sirt1) related diseases by inhibition of natural antisense transcript to sirtuin 1 - Google Patents
Treatment of sirtuin 1 (sirt1) related diseases by inhibition of natural antisense transcript to sirtuin 1 Download PDFInfo
- Publication number
- CA2746001C CA2746001C CA2746001A CA2746001A CA2746001C CA 2746001 C CA2746001 C CA 2746001C CA 2746001 A CA2746001 A CA 2746001A CA 2746001 A CA2746001 A CA 2746001A CA 2746001 C CA2746001 C CA 2746001C
- Authority
- CA
- Canada
- Prior art keywords
- oligonucleotide
- nucleic acid
- sirt1
- antisense
- sirtuin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010041191 Sirtuin 1 Proteins 0.000 title claims abstract description 186
- 102000000344 Sirtuin 1 Human genes 0.000 title claims abstract description 33
- 108020005544 Antisense RNA Proteins 0.000 title claims abstract description 20
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title abstract description 38
- 201000010099 disease Diseases 0.000 title abstract description 33
- 238000011282 treatment Methods 0.000 title description 54
- 230000005764 inhibitory process Effects 0.000 title description 7
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 250
- 230000014509 gene expression Effects 0.000 claims abstract description 104
- 238000000034 method Methods 0.000 claims abstract description 66
- 150000007523 nucleic acids Chemical group 0.000 claims description 153
- 125000003729 nucleotide group Chemical group 0.000 claims description 133
- 102000039446 nucleic acids Human genes 0.000 claims description 117
- 108020004707 nucleic acids Proteins 0.000 claims description 117
- 239000002773 nucleotide Substances 0.000 claims description 117
- 235000000346 sugar Nutrition 0.000 claims description 37
- -1 phosphate triester Chemical class 0.000 claims description 35
- 230000001965 increasing effect Effects 0.000 claims description 34
- 230000004048 modification Effects 0.000 claims description 32
- 238000012986 modification Methods 0.000 claims description 32
- 230000000295 complement effect Effects 0.000 claims description 30
- 239000003814 drug Substances 0.000 claims description 24
- 108020004459 Small interfering RNA Proteins 0.000 claims description 23
- 108091093037 Peptide nucleic acid Proteins 0.000 claims description 17
- 239000008194 pharmaceutical composition Substances 0.000 claims description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 14
- 238000000338 in vitro Methods 0.000 claims description 11
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 11
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 claims description 6
- 125000005600 alkyl phosphonate group Chemical group 0.000 claims description 4
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 4
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 claims description 4
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 claims 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims 3
- 229910019142 PO4 Inorganic materials 0.000 claims 3
- 125000002619 bicyclic group Chemical group 0.000 claims 3
- 239000010452 phosphate Substances 0.000 claims 3
- 238000004519 manufacturing process Methods 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 156
- 102000040430 polynucleotide Human genes 0.000 abstract description 54
- 108091033319 polynucleotide Proteins 0.000 abstract description 54
- 239000002157 polynucleotide Substances 0.000 abstract description 54
- 230000000692 anti-sense effect Effects 0.000 description 170
- 102100031455 NAD-dependent protein deacetylase sirtuin-1 Human genes 0.000 description 155
- 230000009368 gene silencing by RNA Effects 0.000 description 130
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 126
- 108091030071 RNAI Proteins 0.000 description 113
- 108090000623 proteins and genes Proteins 0.000 description 92
- 108020004999 messenger RNA Proteins 0.000 description 81
- 210000004027 cell Anatomy 0.000 description 66
- 239000000074 antisense oligonucleotide Substances 0.000 description 61
- 238000012230 antisense oligonucleotides Methods 0.000 description 61
- 230000006870 function Effects 0.000 description 61
- 241001465754 Metazoa Species 0.000 description 51
- 108020004414 DNA Proteins 0.000 description 50
- 102000053602 DNA Human genes 0.000 description 50
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 46
- 239000000203 mixture Substances 0.000 description 44
- 102000011990 Sirtuin Human genes 0.000 description 42
- 108050002485 Sirtuin Proteins 0.000 description 42
- 230000000694 effects Effects 0.000 description 32
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 31
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 31
- 230000027455 binding Effects 0.000 description 29
- 241000282693 Cercopithecidae Species 0.000 description 25
- 238000003753 real-time PCR Methods 0.000 description 25
- 241000894007 species Species 0.000 description 24
- 238000009396 hybridization Methods 0.000 description 23
- 210000001519 tissue Anatomy 0.000 description 23
- 238000009472 formulation Methods 0.000 description 20
- 239000002502 liposome Substances 0.000 description 20
- 108091081024 Start codon Proteins 0.000 description 19
- 102000004169 proteins and genes Human genes 0.000 description 19
- 238000012360 testing method Methods 0.000 description 19
- 235000018102 proteins Nutrition 0.000 description 18
- 239000004055 small Interfering RNA Substances 0.000 description 18
- 108090000994 Catalytic RNA Proteins 0.000 description 17
- 102000053642 Catalytic RNA Human genes 0.000 description 17
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 17
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 17
- 230000002255 enzymatic effect Effects 0.000 description 17
- 108091092562 ribozyme Proteins 0.000 description 17
- 150000003839 salts Chemical class 0.000 description 17
- 239000000523 sample Substances 0.000 description 17
- 230000001225 therapeutic effect Effects 0.000 description 17
- 150000002632 lipids Chemical group 0.000 description 16
- 108010058731 nopaline synthase Proteins 0.000 description 16
- 230000014616 translation Effects 0.000 description 16
- 241000282414 Homo sapiens Species 0.000 description 15
- 125000000217 alkyl group Chemical group 0.000 description 15
- 238000011160 research Methods 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 14
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 14
- 229940079593 drug Drugs 0.000 description 14
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 13
- 238000013519 translation Methods 0.000 description 13
- 230000014621 translational initiation Effects 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 238000003556 assay Methods 0.000 description 12
- 238000003776 cleavage reaction Methods 0.000 description 12
- 238000003752 polymerase chain reaction Methods 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 12
- 230000001105 regulatory effect Effects 0.000 description 12
- 230000007017 scission Effects 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 230000008685 targeting Effects 0.000 description 12
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 12
- 239000013598 vector Substances 0.000 description 12
- 108020004705 Codon Proteins 0.000 description 11
- 241000282412 Homo Species 0.000 description 11
- 239000012634 fragment Substances 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 11
- 229930024421 Adenine Natural products 0.000 description 10
- 108091026890 Coding region Proteins 0.000 description 10
- 101710163270 Nuclease Proteins 0.000 description 10
- 229960000643 adenine Drugs 0.000 description 10
- 230000003197 catalytic effect Effects 0.000 description 10
- 239000002299 complementary DNA Substances 0.000 description 10
- 239000000178 monomer Substances 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 239000003184 complementary RNA Substances 0.000 description 9
- 239000003623 enhancer Substances 0.000 description 9
- 238000001727 in vivo Methods 0.000 description 9
- 230000035515 penetration Effects 0.000 description 9
- 108090000765 processed proteins & peptides Proteins 0.000 description 9
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 8
- 108700028369 Alleles Proteins 0.000 description 8
- 241000124008 Mammalia Species 0.000 description 8
- 241000288906 Primates Species 0.000 description 8
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 8
- 239000002253 acid Substances 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 238000003197 gene knockdown Methods 0.000 description 8
- 230000007246 mechanism Effects 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 238000010839 reverse transcription Methods 0.000 description 8
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 7
- 101000654471 Mus musculus NAD-dependent protein deacetylase sirtuin-1 Proteins 0.000 description 7
- 108020005038 Terminator Codon Proteins 0.000 description 7
- 241000700605 Viruses Species 0.000 description 7
- 238000007792 addition Methods 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 230000001413 cellular effect Effects 0.000 description 7
- 229940104302 cytosine Drugs 0.000 description 7
- 235000014113 dietary fatty acids Nutrition 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 229930195729 fatty acid Natural products 0.000 description 7
- 239000000194 fatty acid Substances 0.000 description 7
- 229960002949 fluorouracil Drugs 0.000 description 7
- 229910052739 hydrogen Inorganic materials 0.000 description 7
- 239000001257 hydrogen Substances 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 239000004094 surface-active agent Substances 0.000 description 7
- 238000013518 transcription Methods 0.000 description 7
- 230000035897 transcription Effects 0.000 description 7
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 241000282552 Chlorocebus aethiops Species 0.000 description 6
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 6
- 108700026244 Open Reading Frames Proteins 0.000 description 6
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 6
- 150000001408 amides Chemical class 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 6
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 6
- 238000001574 biopsy Methods 0.000 description 6
- 230000037396 body weight Effects 0.000 description 6
- 230000001419 dependent effect Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 150000004665 fatty acids Chemical class 0.000 description 6
- 238000000099 in vitro assay Methods 0.000 description 6
- 238000001802 infusion Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000002844 melting Methods 0.000 description 6
- 230000008018 melting Effects 0.000 description 6
- 150000004713 phosphodiesters Chemical class 0.000 description 6
- 229920001223 polyethylene glycol Polymers 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 6
- 229940113082 thymine Drugs 0.000 description 6
- 229940035893 uracil Drugs 0.000 description 6
- 239000013603 viral vector Substances 0.000 description 6
- 208000024827 Alzheimer disease Diseases 0.000 description 5
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 5
- 101000654472 Homo sapiens NAD-dependent protein deacetylase sirtuin-1 Proteins 0.000 description 5
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 5
- 108700011259 MicroRNAs Proteins 0.000 description 5
- 208000008589 Obesity Diseases 0.000 description 5
- 239000002202 Polyethylene glycol Substances 0.000 description 5
- 108091027967 Small hairpin RNA Proteins 0.000 description 5
- 238000002105 Southern blotting Methods 0.000 description 5
- 241000251131 Sphyrna Species 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 239000002246 antineoplastic agent Substances 0.000 description 5
- 125000002091 cationic group Chemical group 0.000 description 5
- 230000004700 cellular uptake Effects 0.000 description 5
- 235000012000 cholesterol Nutrition 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 235000005911 diet Nutrition 0.000 description 5
- 230000037213 diet Effects 0.000 description 5
- 208000037765 diseases and disorders Diseases 0.000 description 5
- 208000035475 disorder Diseases 0.000 description 5
- 239000000839 emulsion Substances 0.000 description 5
- 238000001990 intravenous administration Methods 0.000 description 5
- 230000000670 limiting effect Effects 0.000 description 5
- 229960000485 methotrexate Drugs 0.000 description 5
- 239000002679 microRNA Substances 0.000 description 5
- 239000002777 nucleoside Substances 0.000 description 5
- 235000020824 obesity Nutrition 0.000 description 5
- 229920000768 polyamine Polymers 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 230000002441 reversible effect Effects 0.000 description 5
- 230000009870 specific binding Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-(hydroxymethyl)cytosine Chemical compound NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 description 4
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 4
- 206010009944 Colon cancer Diseases 0.000 description 4
- 108091092195 Intron Proteins 0.000 description 4
- 239000012124 Opti-MEM Substances 0.000 description 4
- 239000004952 Polyamide Substances 0.000 description 4
- 108010029485 Protein Isoforms Proteins 0.000 description 4
- 102000001708 Protein Isoforms Human genes 0.000 description 4
- 238000002123 RNA extraction Methods 0.000 description 4
- 238000010802 RNA extraction kit Methods 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 4
- 150000007513 acids Chemical class 0.000 description 4
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 4
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 239000002775 capsule Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 229940127089 cytotoxic agent Drugs 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 238000003633 gene expression assay Methods 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 238000005462 in vivo assay Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 230000002452 interceptive effect Effects 0.000 description 4
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 4
- 230000004770 neurodegeneration Effects 0.000 description 4
- 208000015122 neurodegenerative disease Diseases 0.000 description 4
- 230000009871 nonspecific binding Effects 0.000 description 4
- 150000003904 phospholipids Chemical class 0.000 description 4
- 150000003017 phosphorus Chemical class 0.000 description 4
- 230000004962 physiological condition Effects 0.000 description 4
- 229920002647 polyamide Polymers 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 230000000699 topical effect Effects 0.000 description 4
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 4
- 239000003981 vehicle Substances 0.000 description 4
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 description 3
- OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-UHFFFAOYSA-N 0.000 description 3
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 3
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108091032955 Bacterial small RNA Proteins 0.000 description 3
- 239000004380 Cholic acid Substances 0.000 description 3
- 230000004543 DNA replication Effects 0.000 description 3
- 108700024394 Exon Proteins 0.000 description 3
- 241000282326 Felis catus Species 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 102000000574 RNA-Induced Silencing Complex Human genes 0.000 description 3
- 108010016790 RNA-Induced Silencing Complex Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 125000001931 aliphatic group Chemical group 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000003376 axonal effect Effects 0.000 description 3
- 239000003613 bile acid Substances 0.000 description 3
- 239000003833 bile salt Substances 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 235000019416 cholic acid Nutrition 0.000 description 3
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 3
- 229960002471 cholic acid Drugs 0.000 description 3
- 239000005289 controlled pore glass Substances 0.000 description 3
- 125000000753 cycloalkyl group Chemical group 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical group P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 238000007876 drug discovery Methods 0.000 description 3
- 238000010195 expression analysis Methods 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 125000003827 glycol group Chemical group 0.000 description 3
- 125000005842 heteroatom Chemical group 0.000 description 3
- 125000000623 heterocyclic group Chemical group 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 208000030159 metabolic disease Diseases 0.000 description 3
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 3
- 229950006238 nadide Drugs 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 125000003835 nucleoside group Chemical group 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 208000033808 peripheral neuropathy Diseases 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000002028 premature Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 125000006850 spacer group Chemical group 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-O triethylammonium ion Chemical compound CC[NH+](CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-O 0.000 description 3
- 239000001226 triphosphate Substances 0.000 description 3
- 235000011178 triphosphate Nutrition 0.000 description 3
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 3
- 125000002948 undecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- QGVQZRDQPDLHHV-DPAQBDIFSA-N (3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthrene-3-thiol Chemical compound C1C=C2C[C@@H](S)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 QGVQZRDQPDLHHV-DPAQBDIFSA-N 0.000 description 2
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 2
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 108020005345 3' Untranslated Regions Proteins 0.000 description 2
- MJEQLGCFPLHMNV-UHFFFAOYSA-N 4-amino-1-(hydroxymethyl)pyrimidin-2-one Chemical compound NC=1C=CN(CO)C(=O)N=1 MJEQLGCFPLHMNV-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- LQLQRFGHAALLLE-UHFFFAOYSA-N 5-bromouracil Chemical compound BrC1=CNC(=O)NC1=O LQLQRFGHAALLLE-UHFFFAOYSA-N 0.000 description 2
- ZLAQATDNGLKIEV-UHFFFAOYSA-N 5-methyl-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CC1=CNC(=S)NC1=O ZLAQATDNGLKIEV-UHFFFAOYSA-N 0.000 description 2
- UJBCLAXPPIDQEE-UHFFFAOYSA-N 5-prop-1-ynyl-1h-pyrimidine-2,4-dione Chemical compound CC#CC1=CNC(=O)NC1=O UJBCLAXPPIDQEE-UHFFFAOYSA-N 0.000 description 2
- 208000035657 Abasia Diseases 0.000 description 2
- 108010000700 Acetolactate synthase Proteins 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 108020004491 Antisense DNA Proteins 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 108010006654 Bleomycin Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 2
- 108020004394 Complementary RNA Proteins 0.000 description 2
- 241000938605 Crocodylia Species 0.000 description 2
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 2
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 2
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 2
- 108010066133 D-octopine dehydrogenase Proteins 0.000 description 2
- 108010092160 Dactinomycin Proteins 0.000 description 2
- 206010011906 Death Diseases 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 208000032928 Dyslipidaemia Diseases 0.000 description 2
- 239000012594 Earle’s Balanced Salt Solution Substances 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 108091060211 Expressed sequence tag Proteins 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 208000002705 Glucose Intolerance Diseases 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 208000023105 Huntington disease Diseases 0.000 description 2
- 208000031226 Hyperlipidaemia Diseases 0.000 description 2
- 229930010555 Inosine Natural products 0.000 description 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 2
- 206010022489 Insulin Resistance Diseases 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- 101710203526 Integrase Proteins 0.000 description 2
- 208000017170 Lipid metabolism disease Diseases 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 238000011887 Necropsy Methods 0.000 description 2
- 108091092724 Noncoding DNA Proteins 0.000 description 2
- REYJJPSVUYRZGE-UHFFFAOYSA-N Octadecylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 description 2
- 208000001132 Osteoporosis Diseases 0.000 description 2
- 208000018737 Parkinson disease Diseases 0.000 description 2
- 101100054666 Streptomyces halstedii sch3 gene Proteins 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 description 2
- 108091036066 Three prime untranslated region Proteins 0.000 description 2
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 2
- 230000001594 aberrant effect Effects 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- XVIYCJDWYLJQBG-UHFFFAOYSA-N acetic acid;adamantane Chemical compound CC(O)=O.C1C(C2)CC3CC1CC2C3 XVIYCJDWYLJQBG-UHFFFAOYSA-N 0.000 description 2
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 125000003342 alkenyl group Chemical group 0.000 description 2
- 125000005083 alkoxyalkoxy group Chemical group 0.000 description 2
- 125000002877 alkyl aryl group Chemical group 0.000 description 2
- 125000000304 alkynyl group Chemical group 0.000 description 2
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 2
- 125000005122 aminoalkylamino group Chemical group 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003816 antisense DNA Substances 0.000 description 2
- 125000003710 aryl alkyl group Chemical group 0.000 description 2
- 230000037429 base substitution Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 229960001561 bleomycin Drugs 0.000 description 2
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 108010082025 cyan fluorescent protein Proteins 0.000 description 2
- 125000001995 cyclobutyl group Chemical group [H]C1([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 2
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 229960000640 dactinomycin Drugs 0.000 description 2
- GHVNFZFCNZKVNT-UHFFFAOYSA-N decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000001177 diphosphate Substances 0.000 description 2
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 2
- 235000011180 diphosphates Nutrition 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 208000002854 epidermolysis bullosa simplex superficialis Diseases 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 description 2
- 229960000961 floxuridine Drugs 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 238000002509 fluorescent in situ hybridization Methods 0.000 description 2
- 235000012631 food intake Nutrition 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 229940029575 guanosine Drugs 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 125000000592 heterocycloalkyl group Chemical group 0.000 description 2
- 201000001421 hyperglycemia Diseases 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000000138 intercalating agent Substances 0.000 description 2
- 238000007913 intrathecal administration Methods 0.000 description 2
- 238000007914 intraventricular administration Methods 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 125000005647 linker group Chemical group 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 2
- 239000007758 minimum essential medium Substances 0.000 description 2
- 150000004712 monophosphates Chemical class 0.000 description 2
- 201000006417 multiple sclerosis Diseases 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000016273 neuron death Effects 0.000 description 2
- 201000001119 neuropathy Diseases 0.000 description 2
- 230000007823 neuropathy Effects 0.000 description 2
- 108091027963 non-coding RNA Proteins 0.000 description 2
- 102000042567 non-coding RNA Human genes 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 2
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- ONTNXMBMXUNDBF-UHFFFAOYSA-N pentatriacontane-17,18,19-triol Chemical compound CCCCCCCCCCCCCCCCC(O)C(O)C(O)CCCCCCCCCCCCCCCC ONTNXMBMXUNDBF-UHFFFAOYSA-N 0.000 description 2
- 230000003285 pharmacodynamic effect Effects 0.000 description 2
- RDOWQLZANAYVLL-UHFFFAOYSA-N phenanthridine Chemical compound C1=CC=C2C3=CC=CC=C3C=NC2=C1 RDOWQLZANAYVLL-UHFFFAOYSA-N 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 229920000570 polyether Polymers 0.000 description 2
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 2
- 201000009104 prediabetes syndrome Diseases 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 150000003230 pyrimidines Chemical class 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- 125000006853 reporter group Chemical group 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 229920002477 rna polymer Polymers 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000003196 serial analysis of gene expression Methods 0.000 description 2
- 229910001415 sodium ion Inorganic materials 0.000 description 2
- 230000010473 stable expression Effects 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000002123 temporal effect Effects 0.000 description 2
- 239000002562 thickening agent Substances 0.000 description 2
- 150000003568 thioethers Chemical class 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- WYWHKKSPHMUBEB-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 2
- 238000011200 topical administration Methods 0.000 description 2
- 230000002110 toxicologic effect Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 230000005945 translocation Effects 0.000 description 2
- 125000000876 trifluoromethoxy group Chemical group FC(F)(F)O* 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 210000003501 vero cell Anatomy 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 229940075420 xanthine Drugs 0.000 description 2
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 2
- MDKGKXOCJGEUJW-VIFPVBQESA-N (2s)-2-[4-(thiophene-2-carbonyl)phenyl]propanoic acid Chemical compound C1=CC([C@@H](C(O)=O)C)=CC=C1C(=O)C1=CC=CS1 MDKGKXOCJGEUJW-VIFPVBQESA-N 0.000 description 1
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 description 1
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 1
- LRANPJDWHYRCER-UHFFFAOYSA-N 1,2-diazepine Chemical compound N1C=CC=CC=N1 LRANPJDWHYRCER-UHFFFAOYSA-N 0.000 description 1
- HASUWNAFLUMMFI-UHFFFAOYSA-N 1,7-dihydropyrrolo[2,3-d]pyrimidine-2,4-dione Chemical compound O=C1NC(=O)NC2=C1C=CN2 HASUWNAFLUMMFI-UHFFFAOYSA-N 0.000 description 1
- VSNHCAURESNICA-NJFSPNSNSA-N 1-oxidanylurea Chemical compound N[14C](=O)NO VSNHCAURESNICA-NJFSPNSNSA-N 0.000 description 1
- DBPWSSGDRRHUNT-CEGNMAFCSA-N 17α-hydroxyprogesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)C)(O)[C@@]1(C)CC2 DBPWSSGDRRHUNT-CEGNMAFCSA-N 0.000 description 1
- ZMZGFLUUZLELNE-UHFFFAOYSA-N 2,3,5-triiodobenzoic acid Chemical compound OC(=O)C1=CC(I)=CC(I)=C1I ZMZGFLUUZLELNE-UHFFFAOYSA-N 0.000 description 1
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 1
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 description 1
- VEPOHXYIFQMVHW-XOZOLZJESA-N 2,3-dihydroxybutanedioic acid (2S,3S)-3,4-dimethyl-2-phenylmorpholine Chemical compound OC(C(O)C(O)=O)C(O)=O.C[C@H]1[C@@H](OCCN1C)c1ccccc1 VEPOHXYIFQMVHW-XOZOLZJESA-N 0.000 description 1
- QSHACTSJHMKXTE-UHFFFAOYSA-N 2-(2-aminopropyl)-7h-purin-6-amine Chemical compound CC(N)CC1=NC(N)=C2NC=NC2=N1 QSHACTSJHMKXTE-UHFFFAOYSA-N 0.000 description 1
- PIINGYXNCHTJTF-UHFFFAOYSA-N 2-(2-azaniumylethylamino)acetate Chemical group NCCNCC(O)=O PIINGYXNCHTJTF-UHFFFAOYSA-N 0.000 description 1
- BRLJKBOXIVONAG-UHFFFAOYSA-N 2-[[5-(dimethylamino)naphthalen-1-yl]sulfonyl-methylamino]acetic acid Chemical compound C1=CC=C2C(N(C)C)=CC=CC2=C1S(=O)(=O)N(C)CC(O)=O BRLJKBOXIVONAG-UHFFFAOYSA-N 0.000 description 1
- XQCZBXHVTFVIFE-UHFFFAOYSA-N 2-amino-4-hydroxypyrimidine Chemical compound NC1=NC=CC(O)=N1 XQCZBXHVTFVIFE-UHFFFAOYSA-N 0.000 description 1
- XIFVTSIIYVGRHJ-UHFFFAOYSA-N 2-n,2-n,4-n,4-n,6-n-pentamethyl-1,3,5-triazine-2,4,6-triamine Chemical compound CNC1=NC(N(C)C)=NC(N(C)C)=N1 XIFVTSIIYVGRHJ-UHFFFAOYSA-N 0.000 description 1
- YNFSUOFXEVCDTC-UHFFFAOYSA-N 2-n-methyl-7h-purine-2,6-diamine Chemical compound CNC1=NC(N)=C2NC=NC2=N1 YNFSUOFXEVCDTC-UHFFFAOYSA-N 0.000 description 1
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- NMUSYJAQQFHJEW-UHFFFAOYSA-N 5-Azacytidine Natural products O=C1N=C(N)N=CN1C1C(O)C(O)C(CO)O1 NMUSYJAQQFHJEW-UHFFFAOYSA-N 0.000 description 1
- NMUSYJAQQFHJEW-KVTDHHQDSA-N 5-azacytidine Chemical compound O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NMUSYJAQQFHJEW-KVTDHHQDSA-N 0.000 description 1
- JDBGXEHEIRGOBU-UHFFFAOYSA-N 5-hydroxymethyluracil Chemical compound OCC1=CNC(=O)NC1=O JDBGXEHEIRGOBU-UHFFFAOYSA-N 0.000 description 1
- PLUDYDNNASPOEE-UHFFFAOYSA-N 6-(aziridin-1-yl)-1h-pyrimidin-2-one Chemical compound C1=CNC(=O)N=C1N1CC1 PLUDYDNNASPOEE-UHFFFAOYSA-N 0.000 description 1
- SXQMWXNOYLLRBY-UHFFFAOYSA-N 6-(methylamino)purin-8-one Chemical compound CNC1=NC=NC2=NC(=O)N=C12 SXQMWXNOYLLRBY-UHFFFAOYSA-N 0.000 description 1
- DCPSTSVLRXOYGS-UHFFFAOYSA-N 6-amino-1h-pyrimidine-2-thione Chemical compound NC1=CC=NC(S)=N1 DCPSTSVLRXOYGS-UHFFFAOYSA-N 0.000 description 1
- CKOMXBHMKXXTNW-UHFFFAOYSA-N 6-methyladenine Chemical compound CNC1=NC=NC2=C1N=CN2 CKOMXBHMKXXTNW-UHFFFAOYSA-N 0.000 description 1
- VVIAGPKUTFNRDU-UHFFFAOYSA-N 6S-folinic acid Natural products C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-UHFFFAOYSA-N 0.000 description 1
- LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 description 1
- VKKXEIQIGGPMHT-UHFFFAOYSA-N 7h-purine-2,8-diamine Chemical compound NC1=NC=C2NC(N)=NC2=N1 VKKXEIQIGGPMHT-UHFFFAOYSA-N 0.000 description 1
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 1
- 229960005508 8-azaguanine Drugs 0.000 description 1
- 206010065040 AIDS dementia complex Diseases 0.000 description 1
- 208000007848 Alcoholism Diseases 0.000 description 1
- 102000018619 Apolipoproteins A Human genes 0.000 description 1
- 108010027004 Apolipoproteins A Proteins 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 241000239223 Arachnida Species 0.000 description 1
- 241000796533 Arna Species 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010061666 Autonomic neuropathy Diseases 0.000 description 1
- 241001057184 Axion Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000006373 Bell palsy Diseases 0.000 description 1
- 208000019838 Blood disease Diseases 0.000 description 1
- 208000006386 Bone Resorption Diseases 0.000 description 1
- 208000020084 Bone disease Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- QCMYYKRYFNMIEC-UHFFFAOYSA-N COP(O)=O Chemical class COP(O)=O QCMYYKRYFNMIEC-UHFFFAOYSA-N 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 239000005632 Capric acid (CAS 334-48-5) Substances 0.000 description 1
- 206010007559 Cardiac failure congestive Diseases 0.000 description 1
- 208000031229 Cardiomyopathies Diseases 0.000 description 1
- 208000002177 Cataract Diseases 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- 241000282994 Cervidae Species 0.000 description 1
- 241000867607 Chlorocebus sabaeus Species 0.000 description 1
- JZUFKLXOESDKRF-UHFFFAOYSA-N Chlorothiazide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC2=C1NCNS2(=O)=O JZUFKLXOESDKRF-UHFFFAOYSA-N 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 206010048843 Cytomegalovirus chorioretinitis Diseases 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 1
- GZDFHIJNHHMENY-UHFFFAOYSA-N Dimethyl dicarbonate Chemical compound COC(=O)OC(=O)OC GZDFHIJNHHMENY-UHFFFAOYSA-N 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000792859 Enema Species 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- MPJKWIXIYCLVCU-UHFFFAOYSA-N Folinic acid Natural products NC1=NC2=C(N(C=O)C(CNc3ccc(cc3)C(=O)NC(CCC(=O)O)CC(=O)O)CN2)C(=O)N1 MPJKWIXIYCLVCU-UHFFFAOYSA-N 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 206010018364 Glomerulonephritis Diseases 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 108090001102 Hammerhead ribozyme Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 208000016988 Hemorrhagic Stroke Diseases 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108010070875 Human Immunodeficiency Virus tat Gene Products Proteins 0.000 description 1
- 238000009015 Human TaqMan MicroRNA Assay kit Methods 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 1
- XDXDZDZNSLXDNA-TZNDIEGXSA-N Idarubicin Chemical compound C1[C@H](N)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2C[C@@](O)(C(C)=O)C1 XDXDZDZNSLXDNA-TZNDIEGXSA-N 0.000 description 1
- XDXDZDZNSLXDNA-UHFFFAOYSA-N Idarubicin Natural products C1C(N)C(O)C(C)OC1OC1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2CC(O)(C(C)=O)C1 XDXDZDZNSLXDNA-UHFFFAOYSA-N 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 208000029578 Muscle disease Diseases 0.000 description 1
- 241000282339 Mustela Species 0.000 description 1
- 206010068871 Myotonic dystrophy Diseases 0.000 description 1
- 101710112216 NAD-dependent histone deacetylase SIR2 Proteins 0.000 description 1
- 101710189433 NAD-dependent protein deacetylase SRT1 Proteins 0.000 description 1
- 101710169358 NAD-dependent protein deacetylase sirtuin-1 Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108020003217 Nuclear RNA Proteins 0.000 description 1
- 102000043141 Nuclear RNA Human genes 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 241000282943 Odocoileus Species 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- 206010034620 Peripheral sensory neuropathy Diseases 0.000 description 1
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 description 1
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N Phosphinothricin Natural products CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 239000004721 Polyphenylene oxide Substances 0.000 description 1
- 229920000388 Polyphosphate Polymers 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 101710086015 RNA ligase Proteins 0.000 description 1
- 230000026279 RNA modification Effects 0.000 description 1
- 230000007022 RNA scission Effects 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- QNVSXXGDAPORNA-UHFFFAOYSA-N Resveratrol Natural products OC1=CC=CC(C=CC=2C=C(O)C(O)=CC=2)=C1 QNVSXXGDAPORNA-UHFFFAOYSA-N 0.000 description 1
- 208000017442 Retinal disease Diseases 0.000 description 1
- 206010038923 Retinopathy Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 208000026214 Skeletal muscle atrophy Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- LUKBXSAWLPMMSZ-OWOJBTEDSA-N Trans-resveratrol Chemical compound C1=CC(O)=CC=C1\C=C\C1=CC(O)=CC(O)=C1 LUKBXSAWLPMMSZ-OWOJBTEDSA-N 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 206010046543 Urinary incontinence Diseases 0.000 description 1
- 208000036826 VIIth nerve paralysis Diseases 0.000 description 1
- OIRDTQYFTABQOQ-UHTZMRCNSA-N Vidarabine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@@H]1O OIRDTQYFTABQOQ-UHTZMRCNSA-N 0.000 description 1
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 1
- 235000018936 Vitellaria paradoxa Nutrition 0.000 description 1
- RLXCFCYWFYXTON-JTTSDREOSA-N [(3S,8S,9S,10R,13S,14S,17R)-3-hydroxy-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-16-yl] N-hexylcarbamate Chemical group C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC(OC(=O)NCCCCCC)[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 RLXCFCYWFYXTON-JTTSDREOSA-N 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- 229960004150 aciclovir Drugs 0.000 description 1
- 239000003377 acid catalyst Substances 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 206010064930 age-related macular degeneration Diseases 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 206010001584 alcohol abuse Diseases 0.000 description 1
- 208000025746 alcohol use disease Diseases 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229960000473 altretamine Drugs 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- XCPGHVQEEXUHNC-UHFFFAOYSA-N amsacrine Chemical compound COC1=CC(NS(C)(=O)=O)=CC=C1NC1=C(C=CC=C2)C2=NC2=CC=CC=C12 XCPGHVQEEXUHNC-UHFFFAOYSA-N 0.000 description 1
- 229960001220 amsacrine Drugs 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000000507 anthelmentic effect Effects 0.000 description 1
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
- 150000004056 anthraquinones Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 229940037157 anticorticosteroids Drugs 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 229960002756 azacitidine Drugs 0.000 description 1
- 229940125717 barbiturate Drugs 0.000 description 1
- HNYOPLTXPVRDBG-UHFFFAOYSA-N barbituric acid Chemical compound O=C1CC(=O)NC(=O)N1 HNYOPLTXPVRDBG-UHFFFAOYSA-N 0.000 description 1
- 238000002869 basic local alignment search tool Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008236 biological pathway Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000024279 bone resorption Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 235000020934 caloric restriction Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- IVUMCTKHWDRRMH-UHFFFAOYSA-N carprofen Chemical compound C1=CC(Cl)=C[C]2C3=CC=C(C(C(O)=O)C)C=C3N=C21 IVUMCTKHWDRRMH-UHFFFAOYSA-N 0.000 description 1
- 229960003184 carprofen Drugs 0.000 description 1
- 150000001767 cationic compounds Chemical class 0.000 description 1
- 230000032677 cell aging Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 239000005081 chemiluminescent agent Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 229960002155 chlorothiazide Drugs 0.000 description 1
- 150000001841 cholesterols Chemical class 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 101150018117 cobB gene Proteins 0.000 description 1
- ZPUCINDJVBIVPJ-LJISPDSOSA-N ***e Chemical compound O([C@H]1C[C@@H]2CC[C@@H](N2C)[C@H]1C(=O)OC)C(=O)C1=CC=CC=C1 ZPUCINDJVBIVPJ-LJISPDSOSA-N 0.000 description 1
- 230000006999 cognitive decline Effects 0.000 description 1
- 208000010877 cognitive disease Diseases 0.000 description 1
- 229960001338 colchicine Drugs 0.000 description 1
- 238000003340 combinatorial analysis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000008139 complexing agent Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000012059 conventional drug carrier Substances 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 239000003246 corticosteroid Substances 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 150000004775 coumarins Chemical class 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 208000001763 cytomegalovirus retinitis Diseases 0.000 description 1
- 229960003901 dacarbazine Drugs 0.000 description 1
- 108700007153 dansylsarcosine Proteins 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 229940124447 delivery agent Drugs 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- CFCUWKMKBJTWLW-UHFFFAOYSA-N deoliosyl-3C-alpha-L-digitoxosyl-MTM Natural products CC=1C(O)=C2C(O)=C3C(=O)C(OC4OC(C)C(O)C(OC5OC(C)C(O)C(OC6OC(C)C(O)C(C)(O)C6)C5)C4)C(C(OC)C(=O)C(O)C(C)O)CC3=CC2=CC=1OC(OC(C)C1O)CC1OC1CC(O)C(O)C(C)O1 CFCUWKMKBJTWLW-UHFFFAOYSA-N 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000000368 destabilizing effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 208000033679 diabetic kidney disease Diseases 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- RGLYKWWBQGJZGM-ISLYRVAYSA-N diethylstilbestrol Chemical compound C=1C=C(O)C=CC=1C(/CC)=C(\CC)C1=CC=C(O)C=C1 RGLYKWWBQGJZGM-ISLYRVAYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- BPHQZTVXXXJVHI-UHFFFAOYSA-N dimyristoyl phosphatidylglycerol Chemical compound CCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCCCCCCCC BPHQZTVXXXJVHI-UHFFFAOYSA-N 0.000 description 1
- 229960005160 dimyristoylphosphatidylglycerol Drugs 0.000 description 1
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 description 1
- VUSHQLWDOJFSGF-UHFFFAOYSA-L disodium 3-carboxy-3,5-dihydroxy-5-oxopentanoate chloride Chemical compound [Na+].[Na+].Cl.[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O VUSHQLWDOJFSGF-UHFFFAOYSA-L 0.000 description 1
- BPHQZTVXXXJVHI-AJQTZOPKSA-N ditetradecanoyl phosphatidylglycerol Chemical compound CCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@@H](O)CO)OC(=O)CCCCCCCCCCCCC BPHQZTVXXXJVHI-AJQTZOPKSA-N 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 229940126534 drug product Drugs 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 239000007920 enema Substances 0.000 description 1
- 229940079360 enema for constipation Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 108700004025 env Genes Proteins 0.000 description 1
- 101150030339 env gene Proteins 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 229960001904 epirubicin Drugs 0.000 description 1
- ITSGNOIFAJAQHJ-BMFNZSJVSA-N esorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)C[C@H](C)O1 ITSGNOIFAJAQHJ-BMFNZSJVSA-N 0.000 description 1
- 229950002017 esorubicin Drugs 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229960001395 fenbufen Drugs 0.000 description 1
- ZPAKPRAICRBAOD-UHFFFAOYSA-N fenbufen Chemical compound C1=CC(C(=O)CCC(=O)O)=CC=C1C1=CC=CC=C1 ZPAKPRAICRBAOD-UHFFFAOYSA-N 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- LPEPZBJOKDYZAD-UHFFFAOYSA-N flufenamic acid Chemical compound OC(=O)C1=CC=CC=C1NC1=CC=CC(C(F)(F)F)=C1 LPEPZBJOKDYZAD-UHFFFAOYSA-N 0.000 description 1
- 229960004369 flufenamic acid Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- VVIAGPKUTFNRDU-ABLWVSNPSA-N folinic acid Chemical compound C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-ABLWVSNPSA-N 0.000 description 1
- 235000008191 folinic acid Nutrition 0.000 description 1
- 239000011672 folinic acid Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 229960002963 ganciclovir Drugs 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- 229960005277 gemcitabine Drugs 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 208000018706 hematopoietic system disease Diseases 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- UUVWYPNAQBNQJQ-UHFFFAOYSA-N hexamethylmelamine Chemical compound CN(C)C1=NC(N(C)C)=NC(N(C)C)=N1 UUVWYPNAQBNQJQ-UHFFFAOYSA-N 0.000 description 1
- 102000056482 human SIRT1 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229960002899 hydroxyprogesterone Drugs 0.000 description 1
- 229960001680 ibuprofen Drugs 0.000 description 1
- 229960000908 idarubicin Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000005414 inactive ingredient Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 229910001411 inorganic cation Inorganic materials 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- 244000000053 intestinal parasite Species 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 208000020658 intracerebral hemorrhage Diseases 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 229960004768 irinotecan Drugs 0.000 description 1
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 1
- DRAVOWXCEBXPTN-UHFFFAOYSA-N isoguanine Chemical compound NC1=NC(=O)NC2=C1NC=N2 DRAVOWXCEBXPTN-UHFFFAOYSA-N 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 229960001691 leucovorin Drugs 0.000 description 1
- OJMMVQQUTAEWLP-KIDUDLJLSA-N lincomycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@@H](C)O)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 OJMMVQQUTAEWLP-KIDUDLJLSA-N 0.000 description 1
- 229960005287 lincomycin Drugs 0.000 description 1
- 239000008206 lipophilic material Substances 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000009115 maintenance therapy Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 229960001428 mercaptopurine Drugs 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- CFCUWKMKBJTWLW-BKHRDMLASA-N mithramycin Chemical compound O([C@@H]1C[C@@H](O[C@H](C)[C@H]1O)OC=1C=C2C=C3C[C@H]([C@@H](C(=O)C3=C(O)C2=C(O)C=1C)O[C@@H]1O[C@H](C)[C@@H](O)[C@H](O[C@@H]2O[C@H](C)[C@H](O)[C@H](O[C@@H]3O[C@H](C)[C@@H](O)[C@@](C)(O)C3)C2)C1)[C@H](OC)C(=O)[C@@H](O)[C@@H](C)O)[C@H]1C[C@@H](O)[C@H](O)[C@@H](C)O1 CFCUWKMKBJTWLW-BKHRDMLASA-N 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 1
- 229960001156 mitoxantrone Drugs 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 201000006938 muscular dystrophy Diseases 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000000324 neuroprotective effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000006320 pegylation Effects 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- 229960002340 pentostatin Drugs 0.000 description 1
- FPVKHBSQESCIEP-JQCXWYLXSA-N pentostatin Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC[C@H]2O)=C2N=C1 FPVKHBSQESCIEP-JQCXWYLXSA-N 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 208000027232 peripheral nervous system disease Diseases 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229960002895 phenylbutazone Drugs 0.000 description 1
- VYMDGNCVAMGZFE-UHFFFAOYSA-N phenylbutazonum Chemical compound O=C1C(CCCC)C(=O)N(C=2C=CC=CC=2)N1C1=CC=CC=C1 VYMDGNCVAMGZFE-UHFFFAOYSA-N 0.000 description 1
- 150000008298 phosphoramidates Chemical class 0.000 description 1
- 125000004437 phosphorous atom Chemical group 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229960003171 plicamycin Drugs 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 229960003101 pranoprofen Drugs 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000011809 primate model Methods 0.000 description 1
- CPTBDICYNRMXFX-UHFFFAOYSA-N procarbazine Chemical compound CNNCC1=CC=C(C(=O)NC(C)C)C=C1 CPTBDICYNRMXFX-UHFFFAOYSA-N 0.000 description 1
- 229960000624 procarbazine Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 238000003498 protein array Methods 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 239000000018 receptor agonist Substances 0.000 description 1
- 229940044601 receptor agonist Drugs 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 229940016667 resveratrol Drugs 0.000 description 1
- 235000021283 resveratrol Nutrition 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 210000003752 saphenous vein Anatomy 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- JRPHGDYSKGJTKZ-UHFFFAOYSA-K selenophosphate Chemical compound [O-]P([O-])([O-])=[Se] JRPHGDYSKGJTKZ-UHFFFAOYSA-K 0.000 description 1
- 201000005572 sensory peripheral neuropathy Diseases 0.000 description 1
- 230000009919 sequestration Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 101150084733 sir-2.1 gene Proteins 0.000 description 1
- 101150089009 sir2 gene Proteins 0.000 description 1
- 230000025185 skeletal muscle atrophy Effects 0.000 description 1
- 208000013363 skeletal muscle disease Diseases 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- JJICLMJFIKGAAU-UHFFFAOYSA-M sodium;2-amino-9-(1,3-dihydroxypropan-2-yloxymethyl)purin-6-olate Chemical compound [Na+].NC1=NC([O-])=C2N=CN(COC(CO)CO)C2=N1 JJICLMJFIKGAAU-UHFFFAOYSA-M 0.000 description 1
- RMLUKZWYIKEASN-UHFFFAOYSA-M sodium;2-amino-9-(2-hydroxyethoxymethyl)purin-6-olate Chemical compound [Na+].O=C1[N-]C(N)=NC2=C1N=CN2COCCO RMLUKZWYIKEASN-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 210000004003 subcutaneous fat Anatomy 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000019635 sulfation Effects 0.000 description 1
- 238000005670 sulfation reaction Methods 0.000 description 1
- 150000003456 sulfonamides Chemical group 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 150000003457 sulfones Chemical group 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229960004492 suprofen Drugs 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- NRUKOCRGYNPUPR-QBPJDGROSA-N teniposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@@H](OC[C@H]4O3)C=3SC=CC=3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 NRUKOCRGYNPUPR-QBPJDGROSA-N 0.000 description 1
- 229960001278 teniposide Drugs 0.000 description 1
- 229960003604 testosterone Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229960003087 tioguanine Drugs 0.000 description 1
- 229960000303 topotecan Drugs 0.000 description 1
- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 1
- 231100000759 toxicological effect Toxicity 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000000179 transient infrared spectroscopy Methods 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 229960001099 trimetrexate Drugs 0.000 description 1
- NOYPYLRCIDNJJB-UHFFFAOYSA-O trimetrexate Chemical compound COC1=C(OC)C(OC)=CC(NCC=2C(=C3C(N)=[NH+]C(N)=NC3=CC=2)C)=C1 NOYPYLRCIDNJJB-UHFFFAOYSA-O 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 230000036269 ulceration Effects 0.000 description 1
- 210000001113 umbilicus Anatomy 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 229960003636 vidarabine Drugs 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 229960005080 warfarin Drugs 0.000 description 1
- PJVWKTKQMONHTI-UHFFFAOYSA-N warfarin Chemical compound OC=1C2=CC=CC=C2OC(=O)C=1C(CC(=O)C)C1=CC=CC=C1 PJVWKTKQMONHTI-UHFFFAOYSA-N 0.000 description 1
- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 1
- 229960001600 xylazine Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/12—Ophthalmic agents for cataracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01098—Histone deacetylase (3.5.1.98), i.e. sirtuin deacetylase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
- Y10T436/143333—Saccharide [e.g., DNA, etc.]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Neurology (AREA)
- Biotechnology (AREA)
- Physical Education & Sports Medicine (AREA)
- Neurosurgery (AREA)
- Diabetes (AREA)
- Virology (AREA)
- Rheumatology (AREA)
- Hematology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Obesity (AREA)
- Physics & Mathematics (AREA)
- Epidemiology (AREA)
- Urology & Nephrology (AREA)
- Ophthalmology & Optometry (AREA)
- Psychology (AREA)
Abstract
Oligonucleotide compounds modulate expression and/or function of Sirtuin 1(SIRT1) polynucleotides and encoded products thereof. Methods for treating diseases associated with Sirtuin 1(SIRT1) comprise administering one or more Oligonucleotide compounds designed to inhibit the SIRT1 natural antisense transcript to patients.
Description
TREATMENT OF S1RTUIN 1 (SIRTI) RELATED DISEASES BY
INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO SIRTUIN I
CROSS REFERENCE
100011 This application claims the benefit of U.S. Provisional Application No.
61/119.965, filed December 4, 2008, and U.S. Provisional Application No. 61/157,255, tiled March 4, 2009, and U.S.
Provisional Application No. 611259,072. filed November 6, 2009.
FIELD OF INVENTION
100021 Embodiments of the invention comprise oligonucleotides modulating expression and/or function of SIRT I and associated molecules.
BACKGROUND OF THE INVENTION
100031 DNA-RNA and RNA-RNA hybridization arc important to many aspects of nucleic acid function including DNA replication, transcription, and translation.
Hybridization is also central to a variety of technologies that either detect a particular nucleic acid or alter its expression. Antisense nucleotides, for example, disrupt gene expression by hybridizing to target RNA, thereby interfering with RNA splicing, transcription, translation, and replication.
Antisense DNA has the added feature that DNA-RNA hybrids serve as a substrate for digestion by ribonuclease H. an activity that is present in most cell types.
Antisense molecules can be delivered into cells, as is the case for oligodeoxynucleotides (ODNs), or they can be expressed from endogenous genes as RNA molecules. The FDA recently approved an antisense drug, VITRAVENETNI (for treatment of cytomegalovirus retinitis), reflecting that antisense has therapeutic utility.
SUMMARY OF THE INVENTION
100041 This Summary is provided to present a summary of the invention to briefly indicate the nature and substance of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.
100051 In one embodiment, the invention provides methods for inhibiting the action of a natural antisensc transcript by using antisense oligonucleotide(s) targeted to any region of the natural antisensc transcript resulting in up-regulation of the corresponding sense gene. It is also contemplated herein that inhibition of the natural antisense transcript can be achieved by siRNA, ribozymes and small molecules, which are considered to be within the scope of the present invention.
- I-L00061 One embodiment provides a method of modulating function and/or expression of an SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within nucleotides 1 to 1028 of SEQ ID NO:3 or nucleotides 1 to 429 of SEQ Ill NO:4, or nucleotides 1 to 156 of SEQ ID
NO:5 or nucleotides 1 to 593 of SEQ ID NO:6 (Figure 11) thereby modulating function and/or expression of the SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro.
[0007] In another preferred embodiment, an oligonucleotide targets a natural antisense sequence of SIRT1 polynucleotides, for example, polynucleotides set forth as SEQ ID NO: 2 to 5, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Examples of antisense oligonucleotides are set forth as SEQ
ID NOS:7 to 48.
[0008] Another embodiment provides a method of modulating function and/or expression of an SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of the antisense of the SIRT1 polynucleotide; thereby modulating function and/or expression of the SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro.
[0009] Another embodiment provides a method of modulating function and/or expression of an SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to an antisense oligonucleotide to an SIRT1 antisense polynucleotide; thereby modulating function and/or expression of the SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro.
[0010] In a preferred embodiment, a composition comprises one or more antisense oligonucleotides which bind to sense and/or antisense SIRT1 polynucleotides.
[0011] In another preferred embodiment, the oligonucleotides comprise one or more modified or substituted nucleotides.
[0012] In another preferred embodiment, the oligonucleotides comprise one or more modified bonds.
[0013] In yet another embodiment, the modified nucleotides comprise modified bases comprising phosphorothioate, methylphosphonate, peptide nucleic acids, or locked nucleic
INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO SIRTUIN I
CROSS REFERENCE
100011 This application claims the benefit of U.S. Provisional Application No.
61/119.965, filed December 4, 2008, and U.S. Provisional Application No. 61/157,255, tiled March 4, 2009, and U.S.
Provisional Application No. 611259,072. filed November 6, 2009.
FIELD OF INVENTION
100021 Embodiments of the invention comprise oligonucleotides modulating expression and/or function of SIRT I and associated molecules.
BACKGROUND OF THE INVENTION
100031 DNA-RNA and RNA-RNA hybridization arc important to many aspects of nucleic acid function including DNA replication, transcription, and translation.
Hybridization is also central to a variety of technologies that either detect a particular nucleic acid or alter its expression. Antisense nucleotides, for example, disrupt gene expression by hybridizing to target RNA, thereby interfering with RNA splicing, transcription, translation, and replication.
Antisense DNA has the added feature that DNA-RNA hybrids serve as a substrate for digestion by ribonuclease H. an activity that is present in most cell types.
Antisense molecules can be delivered into cells, as is the case for oligodeoxynucleotides (ODNs), or they can be expressed from endogenous genes as RNA molecules. The FDA recently approved an antisense drug, VITRAVENETNI (for treatment of cytomegalovirus retinitis), reflecting that antisense has therapeutic utility.
SUMMARY OF THE INVENTION
100041 This Summary is provided to present a summary of the invention to briefly indicate the nature and substance of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.
100051 In one embodiment, the invention provides methods for inhibiting the action of a natural antisensc transcript by using antisense oligonucleotide(s) targeted to any region of the natural antisensc transcript resulting in up-regulation of the corresponding sense gene. It is also contemplated herein that inhibition of the natural antisense transcript can be achieved by siRNA, ribozymes and small molecules, which are considered to be within the scope of the present invention.
- I-L00061 One embodiment provides a method of modulating function and/or expression of an SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within nucleotides 1 to 1028 of SEQ ID NO:3 or nucleotides 1 to 429 of SEQ Ill NO:4, or nucleotides 1 to 156 of SEQ ID
NO:5 or nucleotides 1 to 593 of SEQ ID NO:6 (Figure 11) thereby modulating function and/or expression of the SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro.
[0007] In another preferred embodiment, an oligonucleotide targets a natural antisense sequence of SIRT1 polynucleotides, for example, polynucleotides set forth as SEQ ID NO: 2 to 5, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Examples of antisense oligonucleotides are set forth as SEQ
ID NOS:7 to 48.
[0008] Another embodiment provides a method of modulating function and/or expression of an SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of the antisense of the SIRT1 polynucleotide; thereby modulating function and/or expression of the SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro.
[0009] Another embodiment provides a method of modulating function and/or expression of an SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro comprising contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to an antisense oligonucleotide to an SIRT1 antisense polynucleotide; thereby modulating function and/or expression of the SIRT1 polynucleotide in patient cells or tissues in vivo or in vitro.
[0010] In a preferred embodiment, a composition comprises one or more antisense oligonucleotides which bind to sense and/or antisense SIRT1 polynucleotides.
[0011] In another preferred embodiment, the oligonucleotides comprise one or more modified or substituted nucleotides.
[0012] In another preferred embodiment, the oligonucleotides comprise one or more modified bonds.
[0013] In yet another embodiment, the modified nucleotides comprise modified bases comprising phosphorothioate, methylphosphonate, peptide nucleic acids, or locked nucleic
-2-acid (LNA) molecules. Preferably, the modified nucleotides are locked nucleic acid molecules, including a-L-LNA.
[0014] In another preferred embodiment, the oligonucleotides are administered to a patient subcutaneously, intra-muscularly, intra- venously or intra-peritoneally.
[0015] In another preferred embodiment, the oligonucleotides are administered in a pharmaceutical composition. A treatment regimen comprises administering the antisense compounds at least once to patient; however, this treatment can be modified to include multiple doses over a period of time. The treatment can be combined with one or more other types of therapies.
[0016] In another preferred embodiment, the oligonucleotides are encapsulated in a liposome.
[0017] Other aspects are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0020] Figure 1 shows Real time PCR results of oligonucleotides designed to S1RT antisense CV396200. The results show that the levels of the SIRT1 mRNA in HepG2 cells are .. significantly increased 48 h after treatment with one of the siRNAs designed to sirtas (sirtas_5, P=0.01). In the same samples the levels of sirtas RNA were significantly decreased after treatment with sirtas 5, but unchanged after treatment with sirtas_6 and sirtas 7, which also had no effect on the SIRT1 mRNA levels (Fig. 1B). sirtas 5, sirtas_6 and sirtas_7 correspond to SEQ ID NO:30, 31 and 32 respectively.
[0021] Figure 2 shows results tor the ongonucleottde walk across the SIRT
antisense CV396200. Real time PCR results show that the levels of the SIRTI mRNA in HepG2 cells are significantly increased 48 h after treatment with three of the antisense oligonucleotides designed to sirtas. CUR-0292 to CUR-309 correspond to SEQ ID NO:7 to 24 respectively.
[0014] In another preferred embodiment, the oligonucleotides are administered to a patient subcutaneously, intra-muscularly, intra- venously or intra-peritoneally.
[0015] In another preferred embodiment, the oligonucleotides are administered in a pharmaceutical composition. A treatment regimen comprises administering the antisense compounds at least once to patient; however, this treatment can be modified to include multiple doses over a period of time. The treatment can be combined with one or more other types of therapies.
[0016] In another preferred embodiment, the oligonucleotides are encapsulated in a liposome.
[0017] Other aspects are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0020] Figure 1 shows Real time PCR results of oligonucleotides designed to S1RT antisense CV396200. The results show that the levels of the SIRT1 mRNA in HepG2 cells are .. significantly increased 48 h after treatment with one of the siRNAs designed to sirtas (sirtas_5, P=0.01). In the same samples the levels of sirtas RNA were significantly decreased after treatment with sirtas 5, but unchanged after treatment with sirtas_6 and sirtas 7, which also had no effect on the SIRT1 mRNA levels (Fig. 1B). sirtas 5, sirtas_6 and sirtas_7 correspond to SEQ ID NO:30, 31 and 32 respectively.
[0021] Figure 2 shows results tor the ongonucleottde walk across the SIRT
antisense CV396200. Real time PCR results show that the levels of the SIRTI mRNA in HepG2 cells are significantly increased 48 h after treatment with three of the antisense oligonucleotides designed to sirtas. CUR-0292 to CUR-309 correspond to SEQ ID NO:7 to 24 respectively.
-3-[0022] Figure 3 shows results for PS, LNA and 2'0 Me Modified oligonucleotides in HepG2 (FIG 3A) and Vero76 cells (FIG 3B). Real time PCR results show that the levels of the SIRT1 mRNA in HepG2 cells are significantly increased 48 h after treatment with PS, LNA, 2'0 Me and 2'0 Me mixmer designed antisense oligonucleotides to SIRT1 antisense.
Levels of SIRT1 mRNA in Vero cells also increased 48 hours after treatment with PS and LNA
modified antisense oligonucleotides to SIRT1 antisense. Bars denoted as CUR-0245, CUR-963, C1JR-0688, CUR-0740 and CUR-0664 correspond to SEQ 1D :25 to 29 respectively.
[0023] Figure 4 shows PCR results of Monkey Fat Biopsies. Real time PCR
results show an increase in SIRTI mRNA levels in fat biopsies from monkeys dosed with CUR-963, an oligonucleotide designed to SIRT1 antisense CV396200.1. CUR-963 corresponds to SEQ ID
26.
[0024] Figure 5 shows PCR results of primary monkey liver hepatocytes. Real time PCR
results show an increase in SIRT1 mRNA levels after treatment with CUR-0245, an oligonucleotide against SIRT1 antisense. Bar denoted as CUR-0245 corresponds to SEQ ID
25.
[0025] Figure 6 shows results for oligonucleotides designed to SIRT antisense CV396200.
Real Time PCR results show that levels of SIRT1 mRNA in HepG2 cells are significantly increased in one of the oligonucleotides designed to SIRT1 antisense CV396200.
The bars denoted as CUR-1230, CUR-1231, CUR-1232 and CUR-1233 correspond to SEQ ID NOs:
to 36 [0026] Figure 7 shows results for oligonucleotides designed to SIRT antisense CV428275.
Real Time PCR results show that levels of SIRT1 mRNA in HepG2 cells are significantly increased in two of the oligonucleotides designed to SIRT1 antisense CV428275.
The bars denoted as CUR-1302, CUR-1304, CUR-1303 and CUR-1305 correspond to SEQ ID NOs:
to 40.
100271 Figure 8 shows Real time PCR results. The results show that a significant increase in SIRT1 mRNA levels in HepG2 cells 48 hours after treatment with one of the oligonucleotides designed to S1RT antisense BE717453. The bars denoted as CUR-1264, CUR1265 and CUR-1266 correspond to SEQ ID NOs.:41 to 43 respectively.
[0028] Figure 9 shows Real time PCR results. The results show that the levels of the SIRT1 mRNA in HepG2 cells are significantly increased 48 h after treatment with three of the oligonucleotides designed to SIRT1 antisense AV718812. The bars denoted as CUR-1294, CUR-1297, CUR-1295, CUR-1296 and CUR-1298 correspond to SEQ ID NOs.: 44 to 48 respectively.
Levels of SIRT1 mRNA in Vero cells also increased 48 hours after treatment with PS and LNA
modified antisense oligonucleotides to SIRT1 antisense. Bars denoted as CUR-0245, CUR-963, C1JR-0688, CUR-0740 and CUR-0664 correspond to SEQ 1D :25 to 29 respectively.
[0023] Figure 4 shows PCR results of Monkey Fat Biopsies. Real time PCR
results show an increase in SIRTI mRNA levels in fat biopsies from monkeys dosed with CUR-963, an oligonucleotide designed to SIRT1 antisense CV396200.1. CUR-963 corresponds to SEQ ID
26.
[0024] Figure 5 shows PCR results of primary monkey liver hepatocytes. Real time PCR
results show an increase in SIRT1 mRNA levels after treatment with CUR-0245, an oligonucleotide against SIRT1 antisense. Bar denoted as CUR-0245 corresponds to SEQ ID
25.
[0025] Figure 6 shows results for oligonucleotides designed to SIRT antisense CV396200.
Real Time PCR results show that levels of SIRT1 mRNA in HepG2 cells are significantly increased in one of the oligonucleotides designed to SIRT1 antisense CV396200.
The bars denoted as CUR-1230, CUR-1231, CUR-1232 and CUR-1233 correspond to SEQ ID NOs:
to 36 [0026] Figure 7 shows results for oligonucleotides designed to SIRT antisense CV428275.
Real Time PCR results show that levels of SIRT1 mRNA in HepG2 cells are significantly increased in two of the oligonucleotides designed to SIRT1 antisense CV428275.
The bars denoted as CUR-1302, CUR-1304, CUR-1303 and CUR-1305 correspond to SEQ ID NOs:
to 40.
100271 Figure 8 shows Real time PCR results. The results show that a significant increase in SIRT1 mRNA levels in HepG2 cells 48 hours after treatment with one of the oligonucleotides designed to S1RT antisense BE717453. The bars denoted as CUR-1264, CUR1265 and CUR-1266 correspond to SEQ ID NOs.:41 to 43 respectively.
[0028] Figure 9 shows Real time PCR results. The results show that the levels of the SIRT1 mRNA in HepG2 cells are significantly increased 48 h after treatment with three of the oligonucleotides designed to SIRT1 antisense AV718812. The bars denoted as CUR-1294, CUR-1297, CUR-1295, CUR-1296 and CUR-1298 correspond to SEQ ID NOs.: 44 to 48 respectively.
-4-=
[0029] Figure 10 shows SEQ ID NO: 1: Homo sapiens sirtuin (silent mating type information regulation 2 homolog) 1 (S. cerevisiae) (SIRT1), mRNA (NCBI Accession Number:
NM 012238.3) and SEQ ID NO: 2 shows the genomic sequence of SIRT (exons are shown in capital letters, introns in small) [0030] Figure 11 shows:
SEQ ID NO: 3: Expanded natural antisense sequence (CV396200 - expanded) SEQ ID NO:4: Natural Antisense sequence (CV428275) SEQ ID NO: 5: Natural Antisense Sequence (BE717453) SEQ ID NO:6: Natural Antisense Sequence (AV718812) [00311 Figure 12 shows SEQ ID NO: 7 t024,* indicates phosphothioate bond [0032] Figure 13 shows SEQ ID NO: 25 to 29, * indicates phosphothioate bond, +
indicates LNA and m indicates 20 Me [0033] Figure 14 shows SEQ ID NO: 30 to 32, the antisense sequence of the double stranded test oligonucleotides designed to S1RT antisense CV396200 which correspond to sirtas_5, sirtas_6 and sirtas_7 respectively [0034] Figure 15 shows SEQ ID NO: 33, 34, 35 and 36 designed to SIRT1 antisense CV396200.
[0035] Figure 16 shows SEQ ID NO: 37, 38, 39 and 40 designed to SIRT1 antisense CV428275.
10036] Figure 17 shows SEQ ID NO:41, 42 and 43 designed to SI RT1 antisense BE717453.
[0037] Figure 18 shows SEQ ID NO:44, 45, 46, 47 and 48 designed to SIRTI
antisense AV718812.
[0038] Figure 19 shows SEQ ID NO: 50 to 52. SEQ ID NO:49 correspond to the exon 4 of the SIRT1 natural antisense CV396200, SEQ ID NO:50, 51 and 52 corresponding to the forward primer sequence, reverse primer sequence and the reporter sequence respectively.
100391 Figure 20 shows SEQ ID NO: 53 that corresponds to CUR 962, * indicates phosphothioate bond and + indicates LNA.
DETAILED DESCRIPTION OF THE INVENTION
[0040] Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present
[0029] Figure 10 shows SEQ ID NO: 1: Homo sapiens sirtuin (silent mating type information regulation 2 homolog) 1 (S. cerevisiae) (SIRT1), mRNA (NCBI Accession Number:
NM 012238.3) and SEQ ID NO: 2 shows the genomic sequence of SIRT (exons are shown in capital letters, introns in small) [0030] Figure 11 shows:
SEQ ID NO: 3: Expanded natural antisense sequence (CV396200 - expanded) SEQ ID NO:4: Natural Antisense sequence (CV428275) SEQ ID NO: 5: Natural Antisense Sequence (BE717453) SEQ ID NO:6: Natural Antisense Sequence (AV718812) [00311 Figure 12 shows SEQ ID NO: 7 t024,* indicates phosphothioate bond [0032] Figure 13 shows SEQ ID NO: 25 to 29, * indicates phosphothioate bond, +
indicates LNA and m indicates 20 Me [0033] Figure 14 shows SEQ ID NO: 30 to 32, the antisense sequence of the double stranded test oligonucleotides designed to S1RT antisense CV396200 which correspond to sirtas_5, sirtas_6 and sirtas_7 respectively [0034] Figure 15 shows SEQ ID NO: 33, 34, 35 and 36 designed to SIRT1 antisense CV396200.
[0035] Figure 16 shows SEQ ID NO: 37, 38, 39 and 40 designed to SIRT1 antisense CV428275.
10036] Figure 17 shows SEQ ID NO:41, 42 and 43 designed to SI RT1 antisense BE717453.
[0037] Figure 18 shows SEQ ID NO:44, 45, 46, 47 and 48 designed to SIRTI
antisense AV718812.
[0038] Figure 19 shows SEQ ID NO: 50 to 52. SEQ ID NO:49 correspond to the exon 4 of the SIRT1 natural antisense CV396200, SEQ ID NO:50, 51 and 52 corresponding to the forward primer sequence, reverse primer sequence and the reporter sequence respectively.
100391 Figure 20 shows SEQ ID NO: 53 that corresponds to CUR 962, * indicates phosphothioate bond and + indicates LNA.
DETAILED DESCRIPTION OF THE INVENTION
[0040] Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present
-5-invention is not limited by the ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.
[0041] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences arc intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In preferred embodiments, the genes or nucleic acid sequences are human.
Definitions [0042] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof arc used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."
[0043] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on .. how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value.
Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.
[0041] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences arc intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In preferred embodiments, the genes or nucleic acid sequences are human.
Definitions [0042] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof arc used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."
[0043] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on .. how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value.
Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.
-6-[0044] As used herein, the term "mRNA" means the presently known mRNA
transcript(s) of a targeted gene, and any further transcripts which may be elucidated.
[0045] By "antisense oligonucleotides" or "antisense compound" is meant an RNA
or DNA
molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide it binds to another RNA target by means of RNA-RNA
interactions and alters the activity of the target RNA (Eguchi et al., 1991 Ann. Rev. Biochent.
60, 631-652). An antisense oligonucleotide can upregulate or downregulate expression and/or function of a particular polynucleotide. The definition is meant to include any foreign RNA
or DNA
molecule which is useful from a therapeutic, diagnostic, or other viewpoint.
Such molecules include, for example, antisense RNA or DNA molecules, interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.
[0046] In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term "oligonucleotide", also includes linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like. Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
[0047] The oligonucleotide may be "chimeric", that is, composed of different regions. In the context of this invention "chimeric" compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA
region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotides compound. These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties.
The desired properties of the oligonucleotide include, but arc not limited, for example, to increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may
transcript(s) of a targeted gene, and any further transcripts which may be elucidated.
[0045] By "antisense oligonucleotides" or "antisense compound" is meant an RNA
or DNA
molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide it binds to another RNA target by means of RNA-RNA
interactions and alters the activity of the target RNA (Eguchi et al., 1991 Ann. Rev. Biochent.
60, 631-652). An antisense oligonucleotide can upregulate or downregulate expression and/or function of a particular polynucleotide. The definition is meant to include any foreign RNA
or DNA
molecule which is useful from a therapeutic, diagnostic, or other viewpoint.
Such molecules include, for example, antisense RNA or DNA molecules, interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.
[0046] In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term "oligonucleotide", also includes linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like. Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
[0047] The oligonucleotide may be "chimeric", that is, composed of different regions. In the context of this invention "chimeric" compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA
region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotides compound. These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties.
The desired properties of the oligonucleotide include, but arc not limited, for example, to increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may
-7-therefore have different properties. The chimeric oligonucleotides of the present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide analogs as described above.
[0048] The oligonucleotide can be composed of regions that can be linked in "register," that is, when the monomers are linked consecutively, as in native DNA, or linked via spacers. The spacers are intended to constitute a covalent "bridge" between the regions and have in preferred cases a length not exceeding about 100 carbon atoms. The spacers may carry different functionalities, for example, having positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, fluorophors etc.), being .. lipophilic, inducing special secondary structures like, for example, alanine containing peptides that induce alpha-helices.
[0049] As used herein" SIRT1" and "Sirtuin 1" are inclusive of all family members, mutants, alleles, fragments, species, coding and noncoding sequences, sense and antisense polynucleotide strands, etc.
[0050] As used herein "SIRT1" shall refer to Silencing mating type information regulator 2 homolog and is a member of the SIRTuin deacetylase protein family. The amino acid sequence of SIRT1 may be found at Genbank Accession number NP--08509.
SIRT1 is the human homolog of the yeast Sir2 protein and exhibits NAD-dependent deacetylase activity.
[0051] As used herein, the words Sirtuinl, SIRT1, sirtuin, silent mating type information regulation 2 homolog 1, hSIR2, hSIRT1, NAD-dependent deacetylase sirtuin-1, SIR2L1, SIR2-like protein 1, are considered the same in the literature and are used interchangeably in the present application.
[0052] As used herein, the term "oligonucleotide specific for" or "oligonucleotide which targets" refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene. Stability of the complexes and duplexes can be determined by theoretical calculations and/or in vitro assays.
Exemplary assays for determining stability of hybridization complexes and duplexes are described in the Examples below.
[0053] As used herein, the term "target nucleic acid" encompasses DNA, RNA
(comprising premRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA, coding, noncoding sequences, sense or antisense polynucleotides. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of
[0048] The oligonucleotide can be composed of regions that can be linked in "register," that is, when the monomers are linked consecutively, as in native DNA, or linked via spacers. The spacers are intended to constitute a covalent "bridge" between the regions and have in preferred cases a length not exceeding about 100 carbon atoms. The spacers may carry different functionalities, for example, having positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, fluorophors etc.), being .. lipophilic, inducing special secondary structures like, for example, alanine containing peptides that induce alpha-helices.
[0049] As used herein" SIRT1" and "Sirtuin 1" are inclusive of all family members, mutants, alleles, fragments, species, coding and noncoding sequences, sense and antisense polynucleotide strands, etc.
[0050] As used herein "SIRT1" shall refer to Silencing mating type information regulator 2 homolog and is a member of the SIRTuin deacetylase protein family. The amino acid sequence of SIRT1 may be found at Genbank Accession number NP--08509.
SIRT1 is the human homolog of the yeast Sir2 protein and exhibits NAD-dependent deacetylase activity.
[0051] As used herein, the words Sirtuinl, SIRT1, sirtuin, silent mating type information regulation 2 homolog 1, hSIR2, hSIRT1, NAD-dependent deacetylase sirtuin-1, SIR2L1, SIR2-like protein 1, are considered the same in the literature and are used interchangeably in the present application.
[0052] As used herein, the term "oligonucleotide specific for" or "oligonucleotide which targets" refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene. Stability of the complexes and duplexes can be determined by theoretical calculations and/or in vitro assays.
Exemplary assays for determining stability of hybridization complexes and duplexes are described in the Examples below.
[0053] As used herein, the term "target nucleic acid" encompasses DNA, RNA
(comprising premRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA, coding, noncoding sequences, sense or antisense polynucleotides. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of
-8-the nucleic acid. This modulation of function of a target nucleic acid by compounds, which specifically hybridize to it, is generally referred to as "antisense". The functions of DNA to be interfered include, for example, replication and transcription. The functions of RNA to be interfered, include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of an encoded product or oligonucleotides.
[0054] RNA interference "RNAi" is mediated by double stranded RNA (dsRNA) molecules that have sequence-specific homology to their "target" nucleic acid sequences (Caplen, N. J., et al. (2001) Proc. Natl. Acad. Sci. USA 98:9742-9747). In certain embodiments of the present invention, the mediators are 5-25 nucleotide "small interfering" RNA
duplexes (siRNAs). The siRNAs are derived from the processing of dsRNA by an RNase enzyme known as Dicer (Bernstein, E., et al. (2001) Nature 409:363-366). siRNA duplex products are recruited into a multi-protein siRNA complex termed RISC (RNA Induced Silencing Complex). Without wishing to be bound by any particular theory, a RISC is then believed to be guided to a target nucleic acid (suitably mRNA), where the siRNA duplex interacts in a sequence-specific way to mediate cleavage in a catalytic fashion (Bernstein, E., et al. (2001) Nature 409:363-366; Boutla, A., et al. (2001) Cum Biol. 11:1776-1780). Small interfering RNAs that can be used in accordance with the present invention can be synthesized and used according to procedures that are well known in the art and that will be familiar to the ordinarily skilled artisan. Small interfering RNAs for use in the methods of the present invention suitably comprise between about 1 to about 50 nucleotides (nt). In examples of non limiting embodiments, siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.
[0055] Selection of appropriate oligonucleotides is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology.
Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an
[0054] RNA interference "RNAi" is mediated by double stranded RNA (dsRNA) molecules that have sequence-specific homology to their "target" nucleic acid sequences (Caplen, N. J., et al. (2001) Proc. Natl. Acad. Sci. USA 98:9742-9747). In certain embodiments of the present invention, the mediators are 5-25 nucleotide "small interfering" RNA
duplexes (siRNAs). The siRNAs are derived from the processing of dsRNA by an RNase enzyme known as Dicer (Bernstein, E., et al. (2001) Nature 409:363-366). siRNA duplex products are recruited into a multi-protein siRNA complex termed RISC (RNA Induced Silencing Complex). Without wishing to be bound by any particular theory, a RISC is then believed to be guided to a target nucleic acid (suitably mRNA), where the siRNA duplex interacts in a sequence-specific way to mediate cleavage in a catalytic fashion (Bernstein, E., et al. (2001) Nature 409:363-366; Boutla, A., et al. (2001) Cum Biol. 11:1776-1780). Small interfering RNAs that can be used in accordance with the present invention can be synthesized and used according to procedures that are well known in the art and that will be familiar to the ordinarily skilled artisan. Small interfering RNAs for use in the methods of the present invention suitably comprise between about 1 to about 50 nucleotides (nt). In examples of non limiting embodiments, siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.
[0055] Selection of appropriate oligonucleotides is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology.
Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an
-9-approximate measure of identity. These procedures allow the selection of oligonucleotides that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species. One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.
100561 By ''enzymatic RNA" is meant an RNA molecule with enzymatic activity (Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
00571 By "decoy RNA" is meant an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV
trans-activation response (TAR) RNA can act as a "decoy" and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al.
(1990) Cell, 63, 601- 608). This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.
100581 As used herein, the term "monomers" typically indicates monomers linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., from about 3-4, to about several hundreds of monomeric units.
Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, methylphosphornates, phosphoroselenoate, phosphoramidate, and the like, as more fully described below.
00591 The term -nucleotide" covers naturally occurring nucleotides as well as nonnaturally occurring nucleotides. It should be clear to the person skilled in the art that various nucleotides which previously have been considered "non-naturally occurring"
have subsequently been found in nature. Thus, "nucleotides" includes not only the known purine and pyrimidine heterocycles-containing molecules, but also heterocyclic analogues and tautomers thereof. Illustrative examples of other types of nucleotides arc molecules containing adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo- N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N6,N6-ethano-2,6-
100561 By ''enzymatic RNA" is meant an RNA molecule with enzymatic activity (Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
00571 By "decoy RNA" is meant an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV
trans-activation response (TAR) RNA can act as a "decoy" and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al.
(1990) Cell, 63, 601- 608). This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.
100581 As used herein, the term "monomers" typically indicates monomers linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., from about 3-4, to about several hundreds of monomeric units.
Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, methylphosphornates, phosphoroselenoate, phosphoramidate, and the like, as more fully described below.
00591 The term -nucleotide" covers naturally occurring nucleotides as well as nonnaturally occurring nucleotides. It should be clear to the person skilled in the art that various nucleotides which previously have been considered "non-naturally occurring"
have subsequently been found in nature. Thus, "nucleotides" includes not only the known purine and pyrimidine heterocycles-containing molecules, but also heterocyclic analogues and tautomers thereof. Illustrative examples of other types of nucleotides arc molecules containing adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo- N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N6,N6-ethano-2,6-
-10-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-naturally occurring" nucleotides described in Benner et al., U.S. Pat No. 5,432,272.
The term "nucleotide" is intended to cover every and all of these examples as well as analogues and tautomers thereof. Especially interesting nucleotides are those containing adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nucleotides in relation to therapeutic and diagnostic application in humans.
Nucleotides include the natural 2'-deoxy and 2'- hydroxyl sugars, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992) as well as their analogs.
[0060] "Analogs" in reference to nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties (see e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, (1997) Nucl.
Acid.
Res., 25(22), 4429- 4443, Toulme, J.J., (2001) Nature Biotechnology 19:17-18;
Manoharan M., (1999) Biochemica et Biophysica Acta 1489:117-139; Freier S. M., (1997) Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery & Development, 3:
203-213, Herdewin P., (2000) Antisense & Nucleic Acid Drug Dev., 10:297-310); 2'-O, 3'-C-linked [3.2.0] bicycloarabinonucleosides (see e.g. N.K Christiensen., et al, (1998)J.
Am. Chem. Soc., 120: 5458-5463; Prakash TP, Bhat B. (2007) Curr Top Med Chem. 7(7):641-9; Eun Jeong Cho, Joo-Woon Lee, Andrew D. Ellington Applications of Aptamers as Sensors Annual Review of Analytical Chemistry, July 2009, Vol. 2, Pages 241-264. Such analogs include synthetic nucleotides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.
[0061] As used herein, "hybridization" means the pairing of substantially complementary strands of oligomeric compounds. One mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleotides) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleotides which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
[0062] An antisense compound is "specifically hybridizable" when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences
The term "nucleotide" is intended to cover every and all of these examples as well as analogues and tautomers thereof. Especially interesting nucleotides are those containing adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nucleotides in relation to therapeutic and diagnostic application in humans.
Nucleotides include the natural 2'-deoxy and 2'- hydroxyl sugars, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992) as well as their analogs.
[0060] "Analogs" in reference to nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties (see e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, (1997) Nucl.
Acid.
Res., 25(22), 4429- 4443, Toulme, J.J., (2001) Nature Biotechnology 19:17-18;
Manoharan M., (1999) Biochemica et Biophysica Acta 1489:117-139; Freier S. M., (1997) Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery & Development, 3:
203-213, Herdewin P., (2000) Antisense & Nucleic Acid Drug Dev., 10:297-310); 2'-O, 3'-C-linked [3.2.0] bicycloarabinonucleosides (see e.g. N.K Christiensen., et al, (1998)J.
Am. Chem. Soc., 120: 5458-5463; Prakash TP, Bhat B. (2007) Curr Top Med Chem. 7(7):641-9; Eun Jeong Cho, Joo-Woon Lee, Andrew D. Ellington Applications of Aptamers as Sensors Annual Review of Analytical Chemistry, July 2009, Vol. 2, Pages 241-264. Such analogs include synthetic nucleotides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.
[0061] As used herein, "hybridization" means the pairing of substantially complementary strands of oligomeric compounds. One mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleotides) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleotides which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
[0062] An antisense compound is "specifically hybridizable" when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences
-11-under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
[0063] As used herein, the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, "stringent conditions" under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated. In general, stringent hybridization conditions comprise low concentrations (<0.15M) of salts with inorganic cations such as Na++ or K++
(i.e., low ionic strength), temperature higher than 20 C - 25 C. below the Tm of the oligomeric compound:target sequence complex, and the presence of denaturants such as formamide, dimethylformamide, dimethyl sulfoxide, or the detergent sodium dodecyl sulfate (SDS). For example, the hybridization rate decreases 1.1% for each 1%
formamide. An example of a high stringency hybridization condition is 0.1X sodium chloride-sodium citrate buffer (SSC)/0.1% (w/v) SDS at 60 C. for 30 minutes.
[0064] "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides on one or two oligomeric strands. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligomeric compound and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleotides such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.
[0065] It is understood in the art that the sequence of an oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure). The oligomeric compounds of the present invention comprise
[0063] As used herein, the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, "stringent conditions" under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated. In general, stringent hybridization conditions comprise low concentrations (<0.15M) of salts with inorganic cations such as Na++ or K++
(i.e., low ionic strength), temperature higher than 20 C - 25 C. below the Tm of the oligomeric compound:target sequence complex, and the presence of denaturants such as formamide, dimethylformamide, dimethyl sulfoxide, or the detergent sodium dodecyl sulfate (SDS). For example, the hybridization rate decreases 1.1% for each 1%
formamide. An example of a high stringency hybridization condition is 0.1X sodium chloride-sodium citrate buffer (SSC)/0.1% (w/v) SDS at 60 C. for 30 minutes.
[0064] "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides on one or two oligomeric strands. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligomeric compound and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleotides such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.
[0065] It is understood in the art that the sequence of an oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure). The oligomeric compounds of the present invention comprise
-12-at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. As such, an antisense compound which is 18 nucleotides in length having 4 (four) noncomplementary nucleotides which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8%
overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., (1990) J. Mot Biol., 215, 403-410; Zhang and Madden, (1997) Genothe Res., 7, 649-656).
Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., (1981) 2, 482-489).
[0066] As used herein, the term "Thermal Melting Point (Tm)" refers to the temperature, under defined ionic strength, pH, and nucleic acid concentration, at which 50%
of the oligonucleotides complementary to the target sequence hybridize to the target sequence at equilibrium. Typically, stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 C for short oligonucleotides (e.g., 10 to 50 nucleotide).
Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
[0067] As used herein, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
[0068] The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, "allelic," "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., (1990) J. Mot Biol., 215, 403-410; Zhang and Madden, (1997) Genothe Res., 7, 649-656).
Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., (1981) 2, 482-489).
[0066] As used herein, the term "Thermal Melting Point (Tm)" refers to the temperature, under defined ionic strength, pH, and nucleic acid concentration, at which 50%
of the oligonucleotides complementary to the target sequence hybridize to the target sequence at equilibrium. Typically, stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 C for short oligonucleotides (e.g., 10 to 50 nucleotide).
Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
[0067] As used herein, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene.
[0068] The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, "allelic," "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
-13-The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another.
Of particular utility in the invention are variants of wild type gene products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered .. mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
[0069] The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.
[0070] Derivative polynucleotides include nucleic acids subjected to chemical modification, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
Derivatives, e.g., derivative oligonucleotides, may comprise non-naturally-occurring portions, such as altered sugar moieties or inter-sugar linkages. Exemplary among these are phosphorothioate and other sulfur containing species which are known in the art. Derivative nucleic acids may also contain labels, including radionucleotides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.
[0071] A "derivative" polypeptide or peptide is one that is modified, for example, by glycosylation, pegylation, phosphorylation, sulfation, reductionialkylation, acylation, chemical coupling, or mild formalin treatment. A derivative may also be modified to contain a detectable label, either directly or indirectly, including, but not limited to, a radioisotope, fluorescent, and enzyme label.
[0072] As used herein, the term "animal" or "patient" is meant to include, for example, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken, reptiles, fish, insects and arachnids.
[0073] "Mammal" covers warm blooded mammals that arc typically under medical care (e.g., humans and domesticated animals). Examples include feline, canine, equine, bovine, and human, as well as just human.
Of particular utility in the invention are variants of wild type gene products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered .. mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
[0069] The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.
[0070] Derivative polynucleotides include nucleic acids subjected to chemical modification, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
Derivatives, e.g., derivative oligonucleotides, may comprise non-naturally-occurring portions, such as altered sugar moieties or inter-sugar linkages. Exemplary among these are phosphorothioate and other sulfur containing species which are known in the art. Derivative nucleic acids may also contain labels, including radionucleotides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.
[0071] A "derivative" polypeptide or peptide is one that is modified, for example, by glycosylation, pegylation, phosphorylation, sulfation, reductionialkylation, acylation, chemical coupling, or mild formalin treatment. A derivative may also be modified to contain a detectable label, either directly or indirectly, including, but not limited to, a radioisotope, fluorescent, and enzyme label.
[0072] As used herein, the term "animal" or "patient" is meant to include, for example, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken, reptiles, fish, insects and arachnids.
[0073] "Mammal" covers warm blooded mammals that arc typically under medical care (e.g., humans and domesticated animals). Examples include feline, canine, equine, bovine, and human, as well as just human.
-14-[0074] "Treating" or "treatment" covers the treatment of a disease-state in a mammal, and includes: (a) preventing the disease-state from occurring in a mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it;
(b) inhibiting the disease-state, e.g., arresting it development; and/or (c) relieving the disease-state, e.g., causing regression of the disease state until a desired endpoint is reached. Treating also includes the amelioration of a symptom of a disease (e.g., lessen the pain or discomfort), wherein such amelioration may or may not be directly affecting the disease (e.g., cause, transmission, expression, etc.).
[0075] "Neurodegenerative diseases" refers to a wide range of diseases and disorders of the central and peripheral nervous system including, for example, Parkinson's Disease, Huntington's Disease, Alzheimer's Disease, amyotrophic lateral sclerosis (ALS), dementia, multiple sclerosis and other diseases and disorders associated with neuronal cell death.
[0076] "Metabolic diseases" refers to a wide range of diseases and disorders of the endocrine system including, for example, insulin resistance, diabetes, obesity, impaired glucose tolerance, high blood cholesterol, hyperglycemia, dyslipidemia and hyperlipidemia.
Polynucleotide and Oligonucleotide Compositions and Molecules [0077] "SIRT1 protein" refers to a member of the sir2 family of sirtuin deacetylases. In one embodiment, a SIRT1 protein includes yeast Sir2 (GenBank Accession No.
P53685), C.
elegans Sir-2.1 (GenBank Accession No. NP--501912), human SIRT1 (GenBank Accession No. NM.sub,--012238 and NP--036370 (or AF083106)) [0078] SIRT1 "Sirtuins" are proteins that include a SIR2 domain, a domain defined as amino acids sequences that are scored as hits in the Pfam family "SIR2"- PF02146 (attached to the Appendix). This family is referenced in the INTERPRO database as INTERPRO
description (entry IPR003000). To identify the presence of a "SIR2" domain in a protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against the Pfam database of HMMs (e.g., the Pfam database, release 9) using the default parameters.The SIR2 domain is indexed in Pfam as PF02146 and in INTERPRO as INTERPRO description (entry IPR003000). A
description of the Pfam database can be found in "The Pfam Protein Families Database"
Bateman A et al (2002) Nucleic Acids Research 30(1):276-280 and Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol, 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad.
(b) inhibiting the disease-state, e.g., arresting it development; and/or (c) relieving the disease-state, e.g., causing regression of the disease state until a desired endpoint is reached. Treating also includes the amelioration of a symptom of a disease (e.g., lessen the pain or discomfort), wherein such amelioration may or may not be directly affecting the disease (e.g., cause, transmission, expression, etc.).
[0075] "Neurodegenerative diseases" refers to a wide range of diseases and disorders of the central and peripheral nervous system including, for example, Parkinson's Disease, Huntington's Disease, Alzheimer's Disease, amyotrophic lateral sclerosis (ALS), dementia, multiple sclerosis and other diseases and disorders associated with neuronal cell death.
[0076] "Metabolic diseases" refers to a wide range of diseases and disorders of the endocrine system including, for example, insulin resistance, diabetes, obesity, impaired glucose tolerance, high blood cholesterol, hyperglycemia, dyslipidemia and hyperlipidemia.
Polynucleotide and Oligonucleotide Compositions and Molecules [0077] "SIRT1 protein" refers to a member of the sir2 family of sirtuin deacetylases. In one embodiment, a SIRT1 protein includes yeast Sir2 (GenBank Accession No.
P53685), C.
elegans Sir-2.1 (GenBank Accession No. NP--501912), human SIRT1 (GenBank Accession No. NM.sub,--012238 and NP--036370 (or AF083106)) [0078] SIRT1 "Sirtuins" are proteins that include a SIR2 domain, a domain defined as amino acids sequences that are scored as hits in the Pfam family "SIR2"- PF02146 (attached to the Appendix). This family is referenced in the INTERPRO database as INTERPRO
description (entry IPR003000). To identify the presence of a "SIR2" domain in a protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against the Pfam database of HMMs (e.g., the Pfam database, release 9) using the default parameters.The SIR2 domain is indexed in Pfam as PF02146 and in INTERPRO as INTERPRO description (entry IPR003000). A
description of the Pfam database can be found in "The Pfam Protein Families Database"
Bateman A et al (2002) Nucleic Acids Research 30(1):276-280 and Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol, 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad.
-15-Sci. USA 84:4355-4358; Krogh et al. (1994) 1 Mol. Biol. 235:1501-1531; and Stultz et al.
(1993) Protein Sci. 2:305-314.
[0079] Targets: In one embodiment, the targets comprise nucleic acid sequences of Sirtuin 1 (SIRT1), including without limitation sense and/or antisense noncoding and/or coding sequences associated with SIRT1.
100801 In preferred embodiments, antisense oligonucleotides are used to prevent or treat diseases or disorders associated with Sirtuin 1 (SIRT1). The sirtuins (SIRTs) are protein-modifying enzymes that are distributed ubiquitously in all organisms. SIRT1 is a mammalian homologue of yeast nicotinamide-adenine-dinucleotide-dependent deacetylase silent information regulator 2 (known as Sir2), which is the best-characterized SIRT
family member. SIRT1 regulates the physiology of cells of the adipocyte lineage.
Modulators of S1RT1 activity can be used to ameliorate, treat, or prevent diseases and disorders associated with adipose physiology, e.g., obesity, an obesity-related disease, or a fat-related metabolic disorder.
[0081] SIRT1 regulates longevity in several model organisms and is involved in several processes in mammalian cells including cell survival, differentiation, and metabolism. SIRT1 induction, either by SIRT-activating compounds such as resveratrol, or metabolic conditioning associated with caloric restriction, could have neuroprotective qualities and thus delay the neurodegenerative process, thereby promoting longevity (Han SH, (2009) J Clin Neurol. Sep;5(3):120-5.; Michan S, et al. (2007) Biochem J. 404(1): 1-13.).
100821 There are several reports that support an axonal protective role for SIRT1 in the neuronal system. Axonal degeneration is a major morphological characteristic observed in both peripheral neuropathies and neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (Fischer LR, et al. (2004) Exp Neurol 185:232-240;
Stokin GB, et al. (2005) Science 307:1282-1288). Axonal degeneration usually occurs in the early stage in degenerative processes and often precedes or correlates closely with clinical symptoms such as cognitive decline (Yamamoto H, et al. (2007) Mol Endocrinol.
21(8):
1745-1755).
[0083] In a preferred embodiment, the oligonucleotides are specific for polynucleotides of SIRT1, which includes, without limitation noncoding regions. The SIRT1 targets comprise variants of SIRT1; mutants of SIRT1, including SNPs; noncoding sequences of SIRT1;
alleles, fragments and the like. Preferably the oligonucleotide is an antisense RNA molecule.
(1993) Protein Sci. 2:305-314.
[0079] Targets: In one embodiment, the targets comprise nucleic acid sequences of Sirtuin 1 (SIRT1), including without limitation sense and/or antisense noncoding and/or coding sequences associated with SIRT1.
100801 In preferred embodiments, antisense oligonucleotides are used to prevent or treat diseases or disorders associated with Sirtuin 1 (SIRT1). The sirtuins (SIRTs) are protein-modifying enzymes that are distributed ubiquitously in all organisms. SIRT1 is a mammalian homologue of yeast nicotinamide-adenine-dinucleotide-dependent deacetylase silent information regulator 2 (known as Sir2), which is the best-characterized SIRT
family member. SIRT1 regulates the physiology of cells of the adipocyte lineage.
Modulators of S1RT1 activity can be used to ameliorate, treat, or prevent diseases and disorders associated with adipose physiology, e.g., obesity, an obesity-related disease, or a fat-related metabolic disorder.
[0081] SIRT1 regulates longevity in several model organisms and is involved in several processes in mammalian cells including cell survival, differentiation, and metabolism. SIRT1 induction, either by SIRT-activating compounds such as resveratrol, or metabolic conditioning associated with caloric restriction, could have neuroprotective qualities and thus delay the neurodegenerative process, thereby promoting longevity (Han SH, (2009) J Clin Neurol. Sep;5(3):120-5.; Michan S, et al. (2007) Biochem J. 404(1): 1-13.).
100821 There are several reports that support an axonal protective role for SIRT1 in the neuronal system. Axonal degeneration is a major morphological characteristic observed in both peripheral neuropathies and neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (Fischer LR, et al. (2004) Exp Neurol 185:232-240;
Stokin GB, et al. (2005) Science 307:1282-1288). Axonal degeneration usually occurs in the early stage in degenerative processes and often precedes or correlates closely with clinical symptoms such as cognitive decline (Yamamoto H, et al. (2007) Mol Endocrinol.
21(8):
1745-1755).
[0083] In a preferred embodiment, the oligonucleotides are specific for polynucleotides of SIRT1, which includes, without limitation noncoding regions. The SIRT1 targets comprise variants of SIRT1; mutants of SIRT1, including SNPs; noncoding sequences of SIRT1;
alleles, fragments and the like. Preferably the oligonucleotide is an antisense RNA molecule.
-16-[0084] In accordance with embodiments of the invention, the target nucleic acid molecule is not limited to SIRT1 polynucleotides alone but extends to any of the isoforms, receptors, homologs, non-coding regions and the like of SIRT1.
[0085] In another preferred embodiment, an oligonucleotide targets a natural antisense sequence (natural antisense to the coding and non-coding regions) of SIRT1 targets, including, without limitation, variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense RNA or DNA molecule.
[0086] In another preferred embodiment, the oligomeric compounds of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenine, variants may be produced which contain thymidine, guanosine, cytidine or other natural or unnatural nucleotides at this position. This may be done at any of the positions of the antisense compound. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.
[0087] In some embodiments, homology, sequence identity or complementarity, between the antisense compound and target is from about 50% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70%
to about 80%. In some embodiments, homology, sequence identity or complementarity, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.
[0088] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired. Such conditions include, i.e., physiological conditions in the case of in vivo assays or therapeutic treatment, and conditions in which assays are performed in the case of in vitro assays.
[0089] An antisense compound, whether DNA, RNA, chimeric, substituted etc, is specifically hybridizablc when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarily to avoid non-specific binding of the antisense compound
[0085] In another preferred embodiment, an oligonucleotide targets a natural antisense sequence (natural antisense to the coding and non-coding regions) of SIRT1 targets, including, without limitation, variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense RNA or DNA molecule.
[0086] In another preferred embodiment, the oligomeric compounds of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenine, variants may be produced which contain thymidine, guanosine, cytidine or other natural or unnatural nucleotides at this position. This may be done at any of the positions of the antisense compound. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.
[0087] In some embodiments, homology, sequence identity or complementarity, between the antisense compound and target is from about 50% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70%
to about 80%. In some embodiments, homology, sequence identity or complementarity, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.
[0088] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired. Such conditions include, i.e., physiological conditions in the case of in vivo assays or therapeutic treatment, and conditions in which assays are performed in the case of in vitro assays.
[0089] An antisense compound, whether DNA, RNA, chimeric, substituted etc, is specifically hybridizablc when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarily to avoid non-specific binding of the antisense compound
-17-to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the ease of in vitro assays, under conditions in which the assays are performed.
[0090] In another preferred embodiment, targeting of SIRT I including without limitation, antisense sequences which are identified and expanded, using for example, PCR, hybridization etc., one or more of the sequences set forth as SEQ ID NOS:7 to 48, and the like, modulate the expression or function of SIRT1. In one embodiment, expression or function is up-regulated as compared to a control. In another preferred embodiment, expression or function is down-regulated as compared to a control.
[0091] In another preferred embodiment, oligonucleotides comprise nucleic acid sequences set forth as SEQ ID NOS:7 to 48, including antisense sequences which are identified and expanded, using for example, PCR, hybridization etc. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like.
Examples of modified bonds or intemucleotide linkages comprise phosphorothioate, phosphorodithioate or the like. In another preferred embodiment, the nucleotides comprise a phosphorus derivative. The phosphorus derivative (or modified phosphate group) which may be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention may be a monophosphate, diphosphate, triphosph ate, alkylphosphate, alkanephosphate, phosphorothioate and the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per se, is also known and need not be described here.
[0092] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisensc oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.
[0093] In embodiments of the present invention oligomeric antisense compounds, particularly oligonucleotides, bind to target nucleic acid molecules and modulate the expression and/or function of molecules encoded by a target gene. The functions of DNA to be interfered comprise, for example, replication and transcription. The functions of RNA to be interfered comprise all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA
to yield one or
[0090] In another preferred embodiment, targeting of SIRT I including without limitation, antisense sequences which are identified and expanded, using for example, PCR, hybridization etc., one or more of the sequences set forth as SEQ ID NOS:7 to 48, and the like, modulate the expression or function of SIRT1. In one embodiment, expression or function is up-regulated as compared to a control. In another preferred embodiment, expression or function is down-regulated as compared to a control.
[0091] In another preferred embodiment, oligonucleotides comprise nucleic acid sequences set forth as SEQ ID NOS:7 to 48, including antisense sequences which are identified and expanded, using for example, PCR, hybridization etc. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like.
Examples of modified bonds or intemucleotide linkages comprise phosphorothioate, phosphorodithioate or the like. In another preferred embodiment, the nucleotides comprise a phosphorus derivative. The phosphorus derivative (or modified phosphate group) which may be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention may be a monophosphate, diphosphate, triphosph ate, alkylphosphate, alkanephosphate, phosphorothioate and the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per se, is also known and need not be described here.
[0092] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisensc oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.
[0093] In embodiments of the present invention oligomeric antisense compounds, particularly oligonucleotides, bind to target nucleic acid molecules and modulate the expression and/or function of molecules encoded by a target gene. The functions of DNA to be interfered comprise, for example, replication and transcription. The functions of RNA to be interfered comprise all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA
to yield one or
-18-more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The functions may be up-regulated or inhibited depending on the functions desired.
100941 The antisense compounds, include, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.
100951 Targeting an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated.
This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes Sirtuin (SIRT I ).
IS 100961 The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term "region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. "Segments" arc defined as smaller or sub-portions of regions within a target nucleic acid. "Sites," as used in the present invention, are defined as positions within a target nucleic acid.
100971 In a preferred embodiment, the antisense oligonucleotides bind to the natural antisense sequences of Sinuin 1 (SIRTI ) and modulate the expression andlor function of Sirtuin 1 (SIRT I )(SEQ ID NO: ). Examples of antisense sequences include SEQ ID NO:3 to 6.
100981 In another preferred embodiment, the antisense oligonucleotides bind to one or more segments of Sirtuin I (SIRT1) polynueleotides and modulate the expression andlor function of Sirtuin I (SIRTI). The segments comprise at least five consecutive nucleotides of the Sirtuin 1 (SIRT ) sense or antisense polynucicotides.
100991 In another preferred embodiment, the antisense oligonucleotides arc specific for natural antisense sequences of Sirtuin 1 (SIRT1) wherein binding of the oligonucleotides to the natural antisense sequences of Sirtuin I (SIRTI ) modulate expression and/or function of Sirtuin I (SIRTI ).
100941 The antisense compounds, include, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.
100951 Targeting an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated.
This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes Sirtuin (SIRT I ).
IS 100961 The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term "region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. "Segments" arc defined as smaller or sub-portions of regions within a target nucleic acid. "Sites," as used in the present invention, are defined as positions within a target nucleic acid.
100971 In a preferred embodiment, the antisense oligonucleotides bind to the natural antisense sequences of Sinuin 1 (SIRTI ) and modulate the expression andlor function of Sirtuin 1 (SIRT I )(SEQ ID NO: ). Examples of antisense sequences include SEQ ID NO:3 to 6.
100981 In another preferred embodiment, the antisense oligonucleotides bind to one or more segments of Sirtuin I (SIRT1) polynueleotides and modulate the expression andlor function of Sirtuin I (SIRTI). The segments comprise at least five consecutive nucleotides of the Sirtuin 1 (SIRT ) sense or antisense polynucicotides.
100991 In another preferred embodiment, the antisense oligonucleotides arc specific for natural antisense sequences of Sirtuin 1 (SIRT1) wherein binding of the oligonucleotides to the natural antisense sequences of Sirtuin I (SIRTI ) modulate expression and/or function of Sirtuin I (SIRTI ).
-19-[0099a] It is provided in an embodiment that the oligonucleotide encompassed herein has at least 90%
sequence identity to a complement of the Sirtuin 1 (SIRT1) natural antisense transcript.
- 19a -1001001 In another preferred embodiment, oligonucleotide compounds comprise sequences set forth as SEQ ID NOS:7 to 48, antisense sequences which are identified and expanded, using for example, KR. hybridization etc These oligonticleoticles can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like.
Examples of modified bonds or internucleotide linkages comprise phosphorothioatc, phosphorodithioate or the like. In another preferred embodiment. the nucleotides comprise a phosphorus derivative. The phosphorus derivative (or modified phosphate group) which may be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention may be a monophosphate. diphosphate, triphosphate. alkylphosphate, alkanephosphatc, phosphorothioatc and the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides,per se. is also known and need not be described here.
1001011 Since, as is known in the art, the translation initiation codon is typically 5'-AUG
(in transcribed mRNA molecules:, 5'-ATG in the corresponding DNA molecule).
the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes has a translation initiation codon having the RNA
sequence 5'-GUG, 5'-U1rG or 5'-CUG., and 5'-AUA. 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is .. typically methionine (in cukaryotes) or formylmethionitte (in prokaryotes).
Eukinyotic and prokaryotic genes may have two or more alternative start codons. any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention. "start codon"
and "translation initiation codon" refer to the codon or codons that arc used in vim to initiate translation of an mRNA transcribed from a gene encoding Sirtuin regardless of the sequence(s) of such codons. A translation termination coda') (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5.-UAG and 5'-liGA (the corresponding DNA
sequences are 5'-TAA. 5'- TAG and 5'-TGA. respectively).
1001021 The terms "start codon region" and "translation initiation codon region" refer to a .. portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e.. 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon.
Consequently, the "start
sequence identity to a complement of the Sirtuin 1 (SIRT1) natural antisense transcript.
- 19a -1001001 In another preferred embodiment, oligonucleotide compounds comprise sequences set forth as SEQ ID NOS:7 to 48, antisense sequences which are identified and expanded, using for example, KR. hybridization etc These oligonticleoticles can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like.
Examples of modified bonds or internucleotide linkages comprise phosphorothioatc, phosphorodithioate or the like. In another preferred embodiment. the nucleotides comprise a phosphorus derivative. The phosphorus derivative (or modified phosphate group) which may be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention may be a monophosphate. diphosphate, triphosphate. alkylphosphate, alkanephosphatc, phosphorothioatc and the like. The preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides,per se. is also known and need not be described here.
1001011 Since, as is known in the art, the translation initiation codon is typically 5'-AUG
(in transcribed mRNA molecules:, 5'-ATG in the corresponding DNA molecule).
the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes has a translation initiation codon having the RNA
sequence 5'-GUG, 5'-U1rG or 5'-CUG., and 5'-AUA. 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is .. typically methionine (in cukaryotes) or formylmethionitte (in prokaryotes).
Eukinyotic and prokaryotic genes may have two or more alternative start codons. any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention. "start codon"
and "translation initiation codon" refer to the codon or codons that arc used in vim to initiate translation of an mRNA transcribed from a gene encoding Sirtuin regardless of the sequence(s) of such codons. A translation termination coda') (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5.-UAG and 5'-liGA (the corresponding DNA
sequences are 5'-TAA. 5'- TAG and 5'-TGA. respectively).
1001021 The terms "start codon region" and "translation initiation codon region" refer to a .. portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e.. 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon.
Consequently, the "start
-20-codon region" (or "translation initiation codon region") and the "stop codon region" (or "translation termination codon region") are all regions that may be targeted effectively with the antisense compounds of the present invention.
[00103] The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a targeted region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.
[00104] Another target region includes the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene). Still another target region includes the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA (or corresponding nucleotides on the gene). The 5' cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site. Another target region for this invention is the 5' cap region.
-- [00105] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. In one embodiment, targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, is particularly useful in situations -- where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. An aberrant fusion junction due to rearrangement or deletion is another embodiment of a target site. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". Introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.
[00106] In another preferred embodiment, the antisense oligonucleotides bind to coding and/or non-coding regions of a target polynueleotide and modulate the expression and/or function of the target molecule.
[00103] The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a targeted region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.
[00104] Another target region includes the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene). Still another target region includes the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA (or corresponding nucleotides on the gene). The 5' cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site. Another target region for this invention is the 5' cap region.
-- [00105] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. In one embodiment, targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, is particularly useful in situations -- where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. An aberrant fusion junction due to rearrangement or deletion is another embodiment of a target site. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". Introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.
[00106] In another preferred embodiment, the antisense oligonucleotides bind to coding and/or non-coding regions of a target polynueleotide and modulate the expression and/or function of the target molecule.
-21-[00107] In another preferred embodiment, the antisense oligonucleotides bind to natural antisense polynucleotides and modulate the expression and/or function of the target molecule.
[00108] In another preferred embodiment, the antisense oligonucleotides bind to sense polynucleotides and modulate the expression and/or function of the target molecule.
-- [00109] Alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants". More specifically, "pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.
[00110] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA
variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants". If no splicing of the pre-mRNA variant occurs then the pre--- mRNA variant is identical to the mRNA variant.
[00111] Variants can be produced through the use of alternative signals to start or stop transcription. Pre-mRNAs and mRNAs can possess more than one start codon or stop codon.
Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use -- an alternative stop codon are known as "alternative stop variants" of that pre-mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also -- embodiments of target nucleic acids.
[00112] The locations on the target nucleic acid to which the antisense compounds hybridize are defined as at least a 5-nucleobase portion of a target region to which an active antisense compound is targeted.
[00113] While the specific sequences of certain exemplary target segments are set forth -- herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional target segments are readily identifiable by one having ordinary skill in the art in view of this disclosure.
[00108] In another preferred embodiment, the antisense oligonucleotides bind to sense polynucleotides and modulate the expression and/or function of the target molecule.
-- [00109] Alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants". More specifically, "pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.
[00110] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA
variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants". If no splicing of the pre-mRNA variant occurs then the pre--- mRNA variant is identical to the mRNA variant.
[00111] Variants can be produced through the use of alternative signals to start or stop transcription. Pre-mRNAs and mRNAs can possess more than one start codon or stop codon.
Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use -- an alternative stop codon are known as "alternative stop variants" of that pre-mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also -- embodiments of target nucleic acids.
[00112] The locations on the target nucleic acid to which the antisense compounds hybridize are defined as at least a 5-nucleobase portion of a target region to which an active antisense compound is targeted.
[00113] While the specific sequences of certain exemplary target segments are set forth -- herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional target segments are readily identifiable by one having ordinary skill in the art in view of this disclosure.
-22-[00114] Target segments 5-100 nucleotides in length comprising a stretch of at least five (5) consecutive nucleotides selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.
[00115] Target segments can include DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 5'-terminus of one of the illustrative preferred target segments (the remaining nucleotides being a consecutive stretch of the same DNA or RNA
beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleotides). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 3'-terminus of one of the illustrative preferred target segments (the remaining nucleotides being a consecutive stretch of the same DNA or RNA
beginning immediately downstream of the 3'-terminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleotides). One having skill in the art armed with the target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.
[00116] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
[00117] In embodiments of the invention the oligonucleotides bind to an antisense strand of a particular target. The oligonucleotides are at least 5 nucleotides in length and can be synthesized so each oligonucleotide targets overlapping sequences such that oligonucleotides are synthesized to cover the entire length of the target polynucleotide. The targets also include coding as well as non coding regions.
[00118] In one embodiment, it is preferred to target specific nucleic acids by antisense oligonucleotides. Targeting an antisense compound to a particular nucleic acid, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a non coding polynucleotide such as for example, non coding RNA (ncRNA).
[00119] RNAs can be classified into (1) messenger RNAs (mRNAs), which are translated into proteins, and (2) non-protein-coding RNAs (ncRNAs). ncRNAs comprise microRNAs, antisense transcripts and other Transcriptional Units (TU) containing a high density of stop codons and lacking any extensive "Open Reading Frame". Many ncRNAs appear to start from initiation sites in 3' untranslated regions (3'UTRs) of protein-coding loci.
ncRNAs are often
[00115] Target segments can include DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 5'-terminus of one of the illustrative preferred target segments (the remaining nucleotides being a consecutive stretch of the same DNA or RNA
beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleotides). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 3'-terminus of one of the illustrative preferred target segments (the remaining nucleotides being a consecutive stretch of the same DNA or RNA
beginning immediately downstream of the 3'-terminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleotides). One having skill in the art armed with the target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.
[00116] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
[00117] In embodiments of the invention the oligonucleotides bind to an antisense strand of a particular target. The oligonucleotides are at least 5 nucleotides in length and can be synthesized so each oligonucleotide targets overlapping sequences such that oligonucleotides are synthesized to cover the entire length of the target polynucleotide. The targets also include coding as well as non coding regions.
[00118] In one embodiment, it is preferred to target specific nucleic acids by antisense oligonucleotides. Targeting an antisense compound to a particular nucleic acid, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a non coding polynucleotide such as for example, non coding RNA (ncRNA).
[00119] RNAs can be classified into (1) messenger RNAs (mRNAs), which are translated into proteins, and (2) non-protein-coding RNAs (ncRNAs). ncRNAs comprise microRNAs, antisense transcripts and other Transcriptional Units (TU) containing a high density of stop codons and lacking any extensive "Open Reading Frame". Many ncRNAs appear to start from initiation sites in 3' untranslated regions (3'UTRs) of protein-coding loci.
ncRNAs are often
-23-rare and at least half of the ncRNAs that have been sequenced by the FANTOM
consortium seem not to be polyadenylated. Most researchers have for obvious reasons focused on polyadenylated mRNAs that are processed and exported to the cytoplasm.
Recently, it was shown that the set of non-polyadenylated nuclear RNAs may be very large, and that many -- such transcripts arise from so-called intergenic regions (Cheng, J. et al.
(2005) Science 308 (5725), 1149-1154; Kapranov, P. et al. (2005). Genome Res 15 (7), 987-997).
The mechanism by which ncRNAs may regulate gene expression is by base pairing with target transcripts. The RNAs that function by base pairing can be grouped into (1) cis encoded RNAs that are encoded at the same genetic location, but on the opposite strand to the RNAs they act upon -- and therefore display perfect complementarity to their target, and (2) trans-encoded RNAs that arc encoded at a chromosomal location distinct from the RNAs they act upon and generally do not exhibit perfect base-pairing potential with their targets.
[00120] Without wishing to be bound by theory, perturbation of an antisense polynucleotide by the antisense oligonucleotides described herein can alter the expression of the corresponding sense messenger RNAs. However, this regulation can either be discordant (antisense knockdown results in messenger RNA elevation) or concordant (antisense knockdown results in concomitant messenger RNA reduction). In these cases, antisense oligonucleotides can be targeted to overlapping or non-overlapping parts of the antisense transcript resulting in its knockdown or sequestration. Coding as well as non-coding antisense -- can be targeted in an identical manner and that either category is capable of regulating the corresponding sense transcripts ¨ either in a concordant or disconcordant manner. The strategies that are employed in identifying new oligonucleotides for use against a target can be based on the knockdown of antisense RNA transcripts by antisense oligonucleotides or any other means of modulating the desired target.
[00121] Strategy 1: In the case of discordant regulation, knocking down the antisense transcript elevates the expression of the conventional (sense) gene. Should that latter gene encode for a known or putative drug target, then knockdown of its antisense counterpart could conceivably mimic the action of a receptor agonist or an enzyme stimulant.
[00122] Strategy 2: In the case of concordant regulation, one could concomitantly knock down both antisense and sense transcripts and thereby achieve synergistic reduction of the conventional (sense) gene expression. If, for example, an antisense oligonucleotide is used to achieve knockdown, then this strategy can be used to apply one antisense oligonucleotide targeted to the sense transcript and another antisense oligonucleotide to the corresponding
consortium seem not to be polyadenylated. Most researchers have for obvious reasons focused on polyadenylated mRNAs that are processed and exported to the cytoplasm.
Recently, it was shown that the set of non-polyadenylated nuclear RNAs may be very large, and that many -- such transcripts arise from so-called intergenic regions (Cheng, J. et al.
(2005) Science 308 (5725), 1149-1154; Kapranov, P. et al. (2005). Genome Res 15 (7), 987-997).
The mechanism by which ncRNAs may regulate gene expression is by base pairing with target transcripts. The RNAs that function by base pairing can be grouped into (1) cis encoded RNAs that are encoded at the same genetic location, but on the opposite strand to the RNAs they act upon -- and therefore display perfect complementarity to their target, and (2) trans-encoded RNAs that arc encoded at a chromosomal location distinct from the RNAs they act upon and generally do not exhibit perfect base-pairing potential with their targets.
[00120] Without wishing to be bound by theory, perturbation of an antisense polynucleotide by the antisense oligonucleotides described herein can alter the expression of the corresponding sense messenger RNAs. However, this regulation can either be discordant (antisense knockdown results in messenger RNA elevation) or concordant (antisense knockdown results in concomitant messenger RNA reduction). In these cases, antisense oligonucleotides can be targeted to overlapping or non-overlapping parts of the antisense transcript resulting in its knockdown or sequestration. Coding as well as non-coding antisense -- can be targeted in an identical manner and that either category is capable of regulating the corresponding sense transcripts ¨ either in a concordant or disconcordant manner. The strategies that are employed in identifying new oligonucleotides for use against a target can be based on the knockdown of antisense RNA transcripts by antisense oligonucleotides or any other means of modulating the desired target.
[00121] Strategy 1: In the case of discordant regulation, knocking down the antisense transcript elevates the expression of the conventional (sense) gene. Should that latter gene encode for a known or putative drug target, then knockdown of its antisense counterpart could conceivably mimic the action of a receptor agonist or an enzyme stimulant.
[00122] Strategy 2: In the case of concordant regulation, one could concomitantly knock down both antisense and sense transcripts and thereby achieve synergistic reduction of the conventional (sense) gene expression. If, for example, an antisense oligonucleotide is used to achieve knockdown, then this strategy can be used to apply one antisense oligonucleotide targeted to the sense transcript and another antisense oligonucleotide to the corresponding
-24-antisense transcript, or a single energetically symmetric antisense oligonucleotide that simultaneously targets overlapping sense and antisense transcripts.
[00123] According to the present invention, antisense compounds include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA
compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA
compounds, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. As such, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one or more of these.
These compounds may be single-stranded, doublestranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds arc routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched. Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially double-stranded compound or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
The two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5' or 3' terminus producing an extension of single stranded character. The double stranded compounds optionally can include overhangs on the ends. Further modifications can include -- conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines, however, in some embodiments, the gene expression or function is up regulated. When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary RNA strands that base pair in Watson-Crick fashion.
[00124] Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or may work via occupancy-based mechanisms. In general, nucleic acids (including oligonucleotides) may be described as "DNA-like" (i.e., generally having one or
[00123] According to the present invention, antisense compounds include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA
compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA
compounds, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. As such, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one or more of these.
These compounds may be single-stranded, doublestranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds arc routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched. Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially double-stranded compound or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
The two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5' or 3' terminus producing an extension of single stranded character. The double stranded compounds optionally can include overhangs on the ends. Further modifications can include -- conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines, however, in some embodiments, the gene expression or function is up regulated. When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary RNA strands that base pair in Watson-Crick fashion.
[00124] Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or may work via occupancy-based mechanisms. In general, nucleic acids (including oligonucleotides) may be described as "DNA-like" (i.e., generally having one or
-25-more 2'-deoxy sugars and, generally, T rather than U bases) or "RNA-like"
(i.e., generally having one or more 2'- hydroxyl or 21-modified sugars and, generally U rather than T bases).
Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-like structure are -- "DNA-like" and those which have A-formlike structure are "RNA-like." In some (chimeric) embodiments, an antisense compound may contain both A- and B-form regions.
[00125] In another preferred embodiment, the desired oligonucleotides or antisense compounds, comprise at least one of: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA
(RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof [00126] dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa. dsRNAs targeting gene promoters induce -- potent transcriptional activation of associated genes. RNAa was demonstrated in human cells using synthetic dsRNAs, termed "small activating RNAs" (saRNAs). It is currently not known whether RNAa is conserved in other organisms.
[00127] Small double-stranded RNA (dsRNA), such as small interfering RNA
(siRNA) and microRNA (miRNA), have been found to be the trigger of an evolutionary conserved -- mechanism known as RNA interference (RNAi). RNAi invariably leads to gene silencing via remodeling chromatin to thereby suppress transcription, degrading complementary mRNA, or blocking protein translation. However, in instances described in detail in the examples section which follows, oligonucleotides are shown to increase the expression and/or function of the Sirtuin 1 (SIRT1) polynucleotides and encoded products thereof. dsRNAs may also act as small activating RNAs (saRNA). Without wishing to be bound by theory, by targeting sequences in gene promoters, saRNAs would induce target gene expression in a phenomenon referred to as dsRNA-induced transcriptional activation (RNAa).
[00128] In a further embodiment, the "preferred target segments" identified herein may be employed in a screen for additional compounds that modulate the expression of Sirtuin 1 -- (SIRT1) polynucleotides. "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding Sirtuin 1 (SIRT1) and which comprise at least a 5-nucleotide portion that is complementary to a preferred target segment.
The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding Sirtuin 1 (SIRT1) polynucleotides with one or more candidate modulators,
(i.e., generally having one or more 2'- hydroxyl or 21-modified sugars and, generally U rather than T bases).
Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-like structure are -- "DNA-like" and those which have A-formlike structure are "RNA-like." In some (chimeric) embodiments, an antisense compound may contain both A- and B-form regions.
[00125] In another preferred embodiment, the desired oligonucleotides or antisense compounds, comprise at least one of: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA
(RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof [00126] dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa. dsRNAs targeting gene promoters induce -- potent transcriptional activation of associated genes. RNAa was demonstrated in human cells using synthetic dsRNAs, termed "small activating RNAs" (saRNAs). It is currently not known whether RNAa is conserved in other organisms.
[00127] Small double-stranded RNA (dsRNA), such as small interfering RNA
(siRNA) and microRNA (miRNA), have been found to be the trigger of an evolutionary conserved -- mechanism known as RNA interference (RNAi). RNAi invariably leads to gene silencing via remodeling chromatin to thereby suppress transcription, degrading complementary mRNA, or blocking protein translation. However, in instances described in detail in the examples section which follows, oligonucleotides are shown to increase the expression and/or function of the Sirtuin 1 (SIRT1) polynucleotides and encoded products thereof. dsRNAs may also act as small activating RNAs (saRNA). Without wishing to be bound by theory, by targeting sequences in gene promoters, saRNAs would induce target gene expression in a phenomenon referred to as dsRNA-induced transcriptional activation (RNAa).
[00128] In a further embodiment, the "preferred target segments" identified herein may be employed in a screen for additional compounds that modulate the expression of Sirtuin 1 -- (SIRT1) polynucleotides. "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding Sirtuin 1 (SIRT1) and which comprise at least a 5-nucleotide portion that is complementary to a preferred target segment.
The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding Sirtuin 1 (SIRT1) polynucleotides with one or more candidate modulators,
-26-and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding Sirtuin 1 (SIRT1) polynucleotides, e.g.
SEQ ID NOS: 7 to 47. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding Sirtuin 1 (SIRT1) polynucleotides, the modulator may then be employed in further investigative studies of the function of Sirtuin 1 (SIRT1) polynucleotides, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.
[00129] Targeting the antisense sequence preferably modulates the function of the target gene. For example, the SIRT1 gene (accession number NM 012238.3; UCSC genome database). In a preferred embodiment, the target is an antisense polynucleotide of the Sirtuin 1 gene. In a preferred embodiment, an antisense oligonucleotide targets sense and/or natural antisense sequences of Sirtuin I (SIRT1) polynucleotides (e.g. accession number NM_012238.3 Seq ID No. 1, Fig 10), variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense molecule and the targets include coding and noncoding regions of antisense and/or sense SIRT1 polynucicotides.
[00130] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexcd) oligonucleotides.
[00131] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., (1998) Nature, 391, 806-811; Timmons and Fire, (1998) Nature, 395, 854; Timmons et al., (2001) Gene, 263, 103-112; Tabara et al., (1998) Science, 282, 430-431; Montgomery et al., (1998) Proc. Natl. Acad. Sci. USA, 95, 15502-15507;
Tuschl et al., (1999) Genes Dev,, 13, 3191-3197; Elbashir et al., (2001) Nature, 411, 494-498; Elbashir et al., (2001) Genes Dev. 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., (2002) Science, 295, 694-697).
[00132] In a preferred embodiment, an antisense oligonucleotide targets Sirtuin 1 (SIRT1) polynucleotides (e.g. accession number NM_012238.3), variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto.
Preferably the oligonucleotide is an antisense molecule.
SEQ ID NOS: 7 to 47. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding Sirtuin 1 (SIRT1) polynucleotides, the modulator may then be employed in further investigative studies of the function of Sirtuin 1 (SIRT1) polynucleotides, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.
[00129] Targeting the antisense sequence preferably modulates the function of the target gene. For example, the SIRT1 gene (accession number NM 012238.3; UCSC genome database). In a preferred embodiment, the target is an antisense polynucleotide of the Sirtuin 1 gene. In a preferred embodiment, an antisense oligonucleotide targets sense and/or natural antisense sequences of Sirtuin I (SIRT1) polynucleotides (e.g. accession number NM_012238.3 Seq ID No. 1, Fig 10), variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto. Preferably the oligonucleotide is an antisense molecule and the targets include coding and noncoding regions of antisense and/or sense SIRT1 polynucicotides.
[00130] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexcd) oligonucleotides.
[00131] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., (1998) Nature, 391, 806-811; Timmons and Fire, (1998) Nature, 395, 854; Timmons et al., (2001) Gene, 263, 103-112; Tabara et al., (1998) Science, 282, 430-431; Montgomery et al., (1998) Proc. Natl. Acad. Sci. USA, 95, 15502-15507;
Tuschl et al., (1999) Genes Dev,, 13, 3191-3197; Elbashir et al., (2001) Nature, 411, 494-498; Elbashir et al., (2001) Genes Dev. 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., (2002) Science, 295, 694-697).
[00132] In a preferred embodiment, an antisense oligonucleotide targets Sirtuin 1 (SIRT1) polynucleotides (e.g. accession number NM_012238.3), variants, alleles, isoforms, homologs, mutants, derivatives, fragments and complementary sequences thereto.
Preferably the oligonucleotide is an antisense molecule.
-27-1001331 In accordance with embodiments of the invention, the target nucleic acid molecule is not limited to Sirtuin I (SIRT1) alone but extends to any of the isoforms, receptors, homologs and the like of Sirtuin I (SIRT I ) molecules.
1001341 In another preferred embodiment, an oligonucleotide targets a natural antisense sequence of SIRT I polynucleotides, for example, polynucleotides set forth as SEQ ID NO:3 t06, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Examples of antisense oligonucleotides are set forth as SEQ
ID NOS: 7 to 48.
1001351 In one embodiment, the oligonucicotides arc complementary to or bind to nucleic acid sequences of Sirtuin I (SIRTI ) antisense, including without limitation noncoding sense and/or antisense sequences associated with Sirtuin I (SIRT1) polyinicleotides and modulate expression and/or function of Sirtuin I (SIRT1) molecules.
1001361 In another preferred embodiment, the oligonucleotidcs are complementary to or bind to nucleic acid sequences of SIRT I natural antisense, set forth as SEQ
ID NO: 3 to6 and modulate expression and/or function of SIRT I molecules.
1001371 In a preferred embodiment, oligonucleotides comprise sequences of at least 5 consecutive nucleotides of SEQ ID NOS:7 to 48 and modulate expression and/or function of Sirtuin I (SIRT1) molecules.
1001381 The polynucleotide targets comprise SIRT1, including family members thereof, variants of S1RT I: mutants of SIRT I, including SN Ps; noncoding sequences of SIRT I ; alleles of SIRTI; species variants, fragments and the like. Preferably the oligonucleotide is an antisense molecule.
1001391 In another preferred embodiment, the oligonucleotide targeting Sirtuin I (SIRTI ) polynucleotides, comprise: antisense RNA, interference RNA (RNAi). short interfering RNA
(siRNA); micro interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa): or, small activating RNA (saRNA).
1001401 In another preferred embodiment, targeting of Sirtuin I (S ) polynucleotides, e.g. SEQ ID NOS: 7 to 48 modulates the expression or function of these targets. In one embodiment, expression or function is up-regulated as compared to a control.
In another preferred embodiment, expression or function is down-regulated as compared to a control.
1001411 In another preferred embodiment. antiscnse compounds comprise sequences set forth as SEQ ID NOS: 7 to 48. These oligonucleotides can comprise one or more modified nucleotides. shorter or longer fragments, modified bonds and the like.
1001421 In another preferred emboditne.m, SEQ ID NOS: 7 to 48 comprise one or more 1.NA nucleotides.
1001431 The modulation of a desired target nucleic acid can be carried out in several ways known in the art. For example, antisense oligonucleotides, siRNA etc.
Enzymatic nucleic acid molecules (e.g., ribozymes) are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be used, for example, to target virtually any RNA transcript (7.aug ef al., 324, Nature 429 1986; Cecil. 260 .1,4.11,4 3030. 1988: and Jefferics et al., 17 Nucleic Achls .. Research 1371, 1989) 1001441 Because of their sequence-specificity, trans-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease tlisman &
MeSwiggen, (1995) Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, (1995).1.
Med. (hem.
38, 2023-2037). Enzymatic nucleic acid molecules can be designed to cleave specific RNA
targets within the background of' cellular RNA. Such a cleavage event renders the mRNA
non-functional and abrogates protein expression from that RNA. In this manner.
synthesis of a protein associated with a disease state can be selectively inhibited.
1001451 In general, enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to CLII the target RNA. Strategic cleavage of such a target RNA
will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target. it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
1001461 Several approaches such as in vitro selection (evolution) strategies (Orgel, (1979) Proc. R. Soc. London. B 205. 435) have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of' phosphodiester linkages and amide linkages, (Joyce, (1989) Gene, 82, 83-87; Bcaudry tft al., (1992) Science 257. 635-641: Joyce. (1992) Scientffic American 267, 90-97; Breaker et al..
(1994) T1BTECH
12, 268; Bartel et al., (1993) Science 261:1411- 1418; Szostak, (1993) TIRS
17, 89-93; Kumar et al.,(1995) FASEB J.. 9. 1183; Breaker. (1996) Curr. Op. Biotech., 7, 442).
[00147] The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA-cleaving ribozymes for the purpose of regulating gene expression. The hammerhead ribozyme, for example, functions with a catalytic rate (kcat) of about 1 min-1 in the presence of saturating (10 mM) concentrations of Mg2+ cofactor. An artificial "RNA ligase" ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of about 100 min-1. In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min-1.
Finally, replacement of a specific residue within the catalytic core of the hammerhead with certain nucleotide analogues gives modified ribozymes that show as much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can promote chemical transformations with catalytic rates that are significantly greater than those displayed in vitro by most natural self-cleaving ribozymes. It is then possible that the structures of certain selfcleaving ribozymes may be optimized to give maximal catalytic activity, or that entirely new RNA motifs can be made that display significantly faster rates for RNA phosphodiester cleavage.
[00148] Intermolecular cleavage of an RNA substrate by an RNA catalyst that fits the "hammerhead" model was first shown in 1987 (Uhlenbeck, 0. C. (1987) Nature, 328: 596-600). The RNA catalyst was recovered and reacted with multiple RNA molecules, demonstrating that it was truly catalytic.
[00149] Catalytic RNAs designed based on the "hammerhead" motif have been used to cleave specific target sequences by making appropriate base changes in the catalytic RNA to maintain necessary base pairing with the target sequences (Haseloff and Gerlach, (1988) Nature, 334, 585; Walbot and Bruening, (1988) Nature, 334, 196; Uhlenbeck, 0.
C. (1987) Nature, 328: 596-600; Koizumi, M., et al. (1988) FEBS Lett., 228: 228-230).
This has allowed use of the catalytic RNA to cleave specific target sequences and indicates that catalytic RNAs designed according to the "hammerhead" model may possibly cleave specific substrate RNAs in vivo. (see Haseloff and Gerlach, (1988) Nature, 334, 585;
Walbot and Bruening, (1988) Nature, 334, 196; Uhlenbeck, 0. C. (1987) Nature, 328: 596-600).
[00150] RNA interference (RNAi) has become a powerful tool for modulating gene expression in mammals and mammalian cells. This approach requires the delivery of small interfering RNA (siRNA) either as RNA itself or as DNA, using an expression plasmid or virus and the coding sequence for small hairpin RNAs that are processed to siRNAs. This system enables efficient transport of the pre-siRNAs to the cytoplasm where they are active and permit the use of regulated and tissue specific promoters for gene expression.
[00151] In a preferred embodiment, an oligonucleotide or antisense compound comprises an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homolog thereof This term includes oligonucleotides composed of naturally occurring nucleotides, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly.
Such modified or substituted oligonucleotides are often desired over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
[00152] According to the present invention, the oligonucleotides or "antisensc compounds"
include antisense oligonucleotides (e.g. RNA, DNA, mimetic, chimera, analog or homolog thereof), ribozymes, external guide sequence (EGS) oligonucleotides, siRNA
compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA
compounds, saRNA, aRNA, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. As such, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one or more of these.
These compounds may be single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched. Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially double-stranded compound or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
The two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5' or 3' terminus producing an extension of single stranded character. The double stranded compounds optionally can include overhangs on the ends. Further modifications can include conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines (Hammond et al., (1991) Nat. Rev. Genet., 2, 110-119; Matzke et al., (2001) Curr. Opin.
Genet. Dev., 11, 221-227; Sharp, (2001) Genes Dev., 15, 485-490). When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary RNA strands that base pair in Watson-Crick fashion.
[00153] Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or may work via occupancy-based mechanisms. In general, nucleic acids (including oligonucleotides) may be described as "DNA-like" (i.e., generally having one or more 2'-deoxy sugars and, generally, T rather than U bases) or "RNA-like"
(i.e., generally having one or more 2'- hydroxyl or 2'-modified sugars and, generally U rather than T bases).
Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-like structure are "DNA like" and those which have A-formlike structure are "RNA like" In some (chimeric) embodiments, an antisense compound may contain both A- and B-form regions.
[00154] The antisense compounds in accordance with this invention can comprise an antisense portion from about 5 to about 80 nucleotides (i.e. from about 5 to about 80 linked nucleosides) in length. This refers to the length of the antisense strand or portion of the antisense compound. In other words, a single-stranded antisense compound of the invention comprises from 5 to about 80 nucleotides, and a double-stranded antisense compound of the invention (such as a dsRNA, for example) comprises an antisense strand or portion of 5 to about 80 nucleotides in length. One of ordinary skill in the art will appreciate that this comprehends antisense portions of 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length, or any range therewithin.
[00155] In one embodiment, the antisense compounds of the invention have antisense portions of 10 to 50 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies oligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therewithin. In some embodiments, the oligonucleotides arc 15 nucleotides in length.
[00156] In one embodiment, the antisense or oligonucleotide compounds of the invention have antisense portions of 12 or 13 to 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range therewithin.
[00157] In a preferred embodiment, administration of at least one oligonucleotide targeting any one or more polynucleotides of Sirtuin 1 (SIRT1), prevents or treats diseases associated with abnormal expression or function of Sirtuin 1 (SIRT1) polynucleotides and encoded products thereof, or other related diseases. Examples of diseases which can be treated with the antisense oligonucleotides comprise: cancer (e.g., breast cancer, colorectal cancer, CCL, CML, prostate cancer), neurodegenerative diseases (e.g., Alzheimer's Disease (AD), Huntington's disease ALS, Parkinson's disease, Amyotrophic Lateral Sclerosis, Multiple Sclerosis, and disorders caused by polyglutaminc aggregation); skeletal muscle disease (e.g., Duchene muscular dystrophy, skeletal muscle atrophy, Becker's dystrophy, or myotonic dystrophy); metabolic diseases (e.g., insulin resistance, diabetes, obesity, impaired glucose tolerance, high blood cholesterol, hyperglycemia, dyslipidemia and hyperlipidemia); adult-onset diabetes, diabetic nephropathy, neuropathy (e.g., sensory neuropathy, autonomic neuropathy, motor neuropathy, retinopathy); bone disease (e.g., osteoporosis), a blood disease (e.g., a leukemia); liver disease (e.g., due to alcohol abuse or hepatitis);
obesity; bone resorption, age-related macular degeneration, AIDS related dementia, ALS, Bell's Palsy, atherosclerosis, cardiac diseases (e.g., cardiac dysrhymias, chronic congestive heart failure, ischemic stroke, coronary artery disease and cardiomyopathy), chronically degenerative disease (e.g., cardiac muscle disease), chronic renal failure, type 2 diabetes, ulceration, cataract, presbiopia, glomerulonephritis, Guillan-Barre syndrome, hemorrhagic stroke, rheumatoid arthritis, inflammatory bowel disease, SLE, Crohn's disease, osteoarthritis, osteoporosis, Chronic Obstructive Pulmonary Disease (COPD), pneumonia, skin aging, and urinary incontinence. Other examples include diseases and disorders associated with neuronal cell death, aging or other condition characterized by unwanted cell loss.
[00158] In another preferred embodiment, the oligomeric compounds of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenosine, variants may be produced which contain thymidine, guanosine or cytidine at this position.
This may be done at any of the positions of the antisense or dsRNA compounds.
These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.
1001591 In some embodiments, homology, sequence identity or complementarity, between the antisense compound and target is from about 40% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70%
to about 3 80%. In some embodiments, homology, sequence identity or complementarily, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%. about 92%, about 94%. about 95%, about 96%. about 97%. about 98%, about 99% or about 100%.
1001601 In another preferred embodiment, the antisense oligonticleotides.
such as for example, nucleic acid molecules set forth in SEQ ID NOS: 7 to 48 comprise one or more substitutions or modifications. In one embodiment, the nucleotides are substituted with locked nucleic acids (LNA).
1001611 In another preferred embodiment, the oligonucleotides target one or more regions of the nucleic acid molecules sense and/or antisense of coding and/or non-coding sequences IS associated with SIRTI and the sequences set forth as SEQ ID NOS: 1 to 6.
The oligonucleotides are also targeted to overlapping regions of SEQ ID NOS: I to 6.
1001621 Certain preferred oligonucleotides of this invention are chimeric oligonucleotides.
"Chimeric oligonucleotides" or "chimeras." in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA
hybrids.
By way of example, RNase H is a cellular endonuelease which cleaves the RNA
strand of an RNA :DNA duplex. Activation of RNitse II, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense modulation of gene expression.
Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used. compared to phosphorothioate deoxyoligonueleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessaty, associated nucleic acid hybridization techniques known in the art. In one preferred embodiment, a chimeric oligonticleotide comprises at least one region modified to increase target binding affinity, and, usually, a region that acts as a substrate for RNAse H. Affinity of an oligontieleotide for its target (in this case, a nucleic acid encoding nis) is routinely determined by measuring the Tilt of an oligonucleotideltarget pair, which is the temperature at which the oligonucleotide and target dissociate;
dissociation is detected spectrophotometrically. The higher the Tin, the greater is the affinity of the oligonucicotide for the target.
1001631 Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides. modified oligonucleotides, oligonucleosides and/or oligonucleotides mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, but arc not limited to, US
patent nos.
5,013,830; 5,149.797; 5, 220,007; 5,256.775; 5,366,878; 5.403311; 5,491,133;
5.565,350:
5.623,065: 5,652.355: 5,652,356; and 5,700.922.
1001641 In another preferred embodiment, the region of the oligonucicotide which is modified comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2.-Oalkyl. 2*-0-alkyl-0-alkyl or 2'-fluoro-modified nucleotide.
In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2' 0-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tai (i.e.. higher target binding affinity) than 2'-deoxyoligonucleotides against a given target. The effect of such increased affinity is to greatly enhance RNAi oligonucicotidc inhibition of gene expression. RNAsc H is a cellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target. and thus can greatly enhance the efficiency of RNAi inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis. In another preferred embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynueleotide. Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides.
A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. Oligonueleotides which contain at least one phosphorothioate modification are -35.
presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance.
Some desirable modifications can be found in De Mesmaeker et al. (1995) Ace. Chem. Res.,
1001341 In another preferred embodiment, an oligonucleotide targets a natural antisense sequence of SIRT I polynucleotides, for example, polynucleotides set forth as SEQ ID NO:3 t06, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto. Examples of antisense oligonucleotides are set forth as SEQ
ID NOS: 7 to 48.
1001351 In one embodiment, the oligonucicotides arc complementary to or bind to nucleic acid sequences of Sirtuin I (SIRTI ) antisense, including without limitation noncoding sense and/or antisense sequences associated with Sirtuin I (SIRT1) polyinicleotides and modulate expression and/or function of Sirtuin I (SIRT1) molecules.
1001361 In another preferred embodiment, the oligonucleotidcs are complementary to or bind to nucleic acid sequences of SIRT I natural antisense, set forth as SEQ
ID NO: 3 to6 and modulate expression and/or function of SIRT I molecules.
1001371 In a preferred embodiment, oligonucleotides comprise sequences of at least 5 consecutive nucleotides of SEQ ID NOS:7 to 48 and modulate expression and/or function of Sirtuin I (SIRT1) molecules.
1001381 The polynucleotide targets comprise SIRT1, including family members thereof, variants of S1RT I: mutants of SIRT I, including SN Ps; noncoding sequences of SIRT I ; alleles of SIRTI; species variants, fragments and the like. Preferably the oligonucleotide is an antisense molecule.
1001391 In another preferred embodiment, the oligonucleotide targeting Sirtuin I (SIRTI ) polynucleotides, comprise: antisense RNA, interference RNA (RNAi). short interfering RNA
(siRNA); micro interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa): or, small activating RNA (saRNA).
1001401 In another preferred embodiment, targeting of Sirtuin I (S ) polynucleotides, e.g. SEQ ID NOS: 7 to 48 modulates the expression or function of these targets. In one embodiment, expression or function is up-regulated as compared to a control.
In another preferred embodiment, expression or function is down-regulated as compared to a control.
1001411 In another preferred embodiment. antiscnse compounds comprise sequences set forth as SEQ ID NOS: 7 to 48. These oligonucleotides can comprise one or more modified nucleotides. shorter or longer fragments, modified bonds and the like.
1001421 In another preferred emboditne.m, SEQ ID NOS: 7 to 48 comprise one or more 1.NA nucleotides.
1001431 The modulation of a desired target nucleic acid can be carried out in several ways known in the art. For example, antisense oligonucleotides, siRNA etc.
Enzymatic nucleic acid molecules (e.g., ribozymes) are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be used, for example, to target virtually any RNA transcript (7.aug ef al., 324, Nature 429 1986; Cecil. 260 .1,4.11,4 3030. 1988: and Jefferics et al., 17 Nucleic Achls .. Research 1371, 1989) 1001441 Because of their sequence-specificity, trans-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for human disease tlisman &
MeSwiggen, (1995) Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, (1995).1.
Med. (hem.
38, 2023-2037). Enzymatic nucleic acid molecules can be designed to cleave specific RNA
targets within the background of' cellular RNA. Such a cleavage event renders the mRNA
non-functional and abrogates protein expression from that RNA. In this manner.
synthesis of a protein associated with a disease state can be selectively inhibited.
1001451 In general, enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to CLII the target RNA. Strategic cleavage of such a target RNA
will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target. it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
1001461 Several approaches such as in vitro selection (evolution) strategies (Orgel, (1979) Proc. R. Soc. London. B 205. 435) have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of' phosphodiester linkages and amide linkages, (Joyce, (1989) Gene, 82, 83-87; Bcaudry tft al., (1992) Science 257. 635-641: Joyce. (1992) Scientffic American 267, 90-97; Breaker et al..
(1994) T1BTECH
12, 268; Bartel et al., (1993) Science 261:1411- 1418; Szostak, (1993) TIRS
17, 89-93; Kumar et al.,(1995) FASEB J.. 9. 1183; Breaker. (1996) Curr. Op. Biotech., 7, 442).
[00147] The development of ribozymes that are optimal for catalytic activity would contribute significantly to any strategy that employs RNA-cleaving ribozymes for the purpose of regulating gene expression. The hammerhead ribozyme, for example, functions with a catalytic rate (kcat) of about 1 min-1 in the presence of saturating (10 mM) concentrations of Mg2+ cofactor. An artificial "RNA ligase" ribozyme has been shown to catalyze the corresponding self-modification reaction with a rate of about 100 min-1. In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min-1.
Finally, replacement of a specific residue within the catalytic core of the hammerhead with certain nucleotide analogues gives modified ribozymes that show as much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can promote chemical transformations with catalytic rates that are significantly greater than those displayed in vitro by most natural self-cleaving ribozymes. It is then possible that the structures of certain selfcleaving ribozymes may be optimized to give maximal catalytic activity, or that entirely new RNA motifs can be made that display significantly faster rates for RNA phosphodiester cleavage.
[00148] Intermolecular cleavage of an RNA substrate by an RNA catalyst that fits the "hammerhead" model was first shown in 1987 (Uhlenbeck, 0. C. (1987) Nature, 328: 596-600). The RNA catalyst was recovered and reacted with multiple RNA molecules, demonstrating that it was truly catalytic.
[00149] Catalytic RNAs designed based on the "hammerhead" motif have been used to cleave specific target sequences by making appropriate base changes in the catalytic RNA to maintain necessary base pairing with the target sequences (Haseloff and Gerlach, (1988) Nature, 334, 585; Walbot and Bruening, (1988) Nature, 334, 196; Uhlenbeck, 0.
C. (1987) Nature, 328: 596-600; Koizumi, M., et al. (1988) FEBS Lett., 228: 228-230).
This has allowed use of the catalytic RNA to cleave specific target sequences and indicates that catalytic RNAs designed according to the "hammerhead" model may possibly cleave specific substrate RNAs in vivo. (see Haseloff and Gerlach, (1988) Nature, 334, 585;
Walbot and Bruening, (1988) Nature, 334, 196; Uhlenbeck, 0. C. (1987) Nature, 328: 596-600).
[00150] RNA interference (RNAi) has become a powerful tool for modulating gene expression in mammals and mammalian cells. This approach requires the delivery of small interfering RNA (siRNA) either as RNA itself or as DNA, using an expression plasmid or virus and the coding sequence for small hairpin RNAs that are processed to siRNAs. This system enables efficient transport of the pre-siRNAs to the cytoplasm where they are active and permit the use of regulated and tissue specific promoters for gene expression.
[00151] In a preferred embodiment, an oligonucleotide or antisense compound comprises an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), or a mimetic, chimera, analog or homolog thereof This term includes oligonucleotides composed of naturally occurring nucleotides, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly.
Such modified or substituted oligonucleotides are often desired over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
[00152] According to the present invention, the oligonucleotides or "antisensc compounds"
include antisense oligonucleotides (e.g. RNA, DNA, mimetic, chimera, analog or homolog thereof), ribozymes, external guide sequence (EGS) oligonucleotides, siRNA
compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA
compounds, saRNA, aRNA, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. As such, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one or more of these.
These compounds may be single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and/or branched. Antisense compounds can include constructs such as, for example, two strands hybridized to form a wholly or partially double-stranded compound or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
The two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5' or 3' terminus producing an extension of single stranded character. The double stranded compounds optionally can include overhangs on the ends. Further modifications can include conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines (Hammond et al., (1991) Nat. Rev. Genet., 2, 110-119; Matzke et al., (2001) Curr. Opin.
Genet. Dev., 11, 221-227; Sharp, (2001) Genes Dev., 15, 485-490). When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary RNA strands that base pair in Watson-Crick fashion.
[00153] Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or may work via occupancy-based mechanisms. In general, nucleic acids (including oligonucleotides) may be described as "DNA-like" (i.e., generally having one or more 2'-deoxy sugars and, generally, T rather than U bases) or "RNA-like"
(i.e., generally having one or more 2'- hydroxyl or 2'-modified sugars and, generally U rather than T bases).
Nucleic acid helices can adopt more than one type of structure, most commonly the A- and B-forms. It is believed that, in general, oligonucleotides which have B-form-like structure are "DNA like" and those which have A-formlike structure are "RNA like" In some (chimeric) embodiments, an antisense compound may contain both A- and B-form regions.
[00154] The antisense compounds in accordance with this invention can comprise an antisense portion from about 5 to about 80 nucleotides (i.e. from about 5 to about 80 linked nucleosides) in length. This refers to the length of the antisense strand or portion of the antisense compound. In other words, a single-stranded antisense compound of the invention comprises from 5 to about 80 nucleotides, and a double-stranded antisense compound of the invention (such as a dsRNA, for example) comprises an antisense strand or portion of 5 to about 80 nucleotides in length. One of ordinary skill in the art will appreciate that this comprehends antisense portions of 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length, or any range therewithin.
[00155] In one embodiment, the antisense compounds of the invention have antisense portions of 10 to 50 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies oligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therewithin. In some embodiments, the oligonucleotides arc 15 nucleotides in length.
[00156] In one embodiment, the antisense or oligonucleotide compounds of the invention have antisense portions of 12 or 13 to 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range therewithin.
[00157] In a preferred embodiment, administration of at least one oligonucleotide targeting any one or more polynucleotides of Sirtuin 1 (SIRT1), prevents or treats diseases associated with abnormal expression or function of Sirtuin 1 (SIRT1) polynucleotides and encoded products thereof, or other related diseases. Examples of diseases which can be treated with the antisense oligonucleotides comprise: cancer (e.g., breast cancer, colorectal cancer, CCL, CML, prostate cancer), neurodegenerative diseases (e.g., Alzheimer's Disease (AD), Huntington's disease ALS, Parkinson's disease, Amyotrophic Lateral Sclerosis, Multiple Sclerosis, and disorders caused by polyglutaminc aggregation); skeletal muscle disease (e.g., Duchene muscular dystrophy, skeletal muscle atrophy, Becker's dystrophy, or myotonic dystrophy); metabolic diseases (e.g., insulin resistance, diabetes, obesity, impaired glucose tolerance, high blood cholesterol, hyperglycemia, dyslipidemia and hyperlipidemia); adult-onset diabetes, diabetic nephropathy, neuropathy (e.g., sensory neuropathy, autonomic neuropathy, motor neuropathy, retinopathy); bone disease (e.g., osteoporosis), a blood disease (e.g., a leukemia); liver disease (e.g., due to alcohol abuse or hepatitis);
obesity; bone resorption, age-related macular degeneration, AIDS related dementia, ALS, Bell's Palsy, atherosclerosis, cardiac diseases (e.g., cardiac dysrhymias, chronic congestive heart failure, ischemic stroke, coronary artery disease and cardiomyopathy), chronically degenerative disease (e.g., cardiac muscle disease), chronic renal failure, type 2 diabetes, ulceration, cataract, presbiopia, glomerulonephritis, Guillan-Barre syndrome, hemorrhagic stroke, rheumatoid arthritis, inflammatory bowel disease, SLE, Crohn's disease, osteoarthritis, osteoporosis, Chronic Obstructive Pulmonary Disease (COPD), pneumonia, skin aging, and urinary incontinence. Other examples include diseases and disorders associated with neuronal cell death, aging or other condition characterized by unwanted cell loss.
[00158] In another preferred embodiment, the oligomeric compounds of the present invention also include variants in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenosine, variants may be produced which contain thymidine, guanosine or cytidine at this position.
This may be done at any of the positions of the antisense or dsRNA compounds.
These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.
1001591 In some embodiments, homology, sequence identity or complementarity, between the antisense compound and target is from about 40% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70%
to about 3 80%. In some embodiments, homology, sequence identity or complementarily, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%. about 92%, about 94%. about 95%, about 96%. about 97%. about 98%, about 99% or about 100%.
1001601 In another preferred embodiment, the antisense oligonticleotides.
such as for example, nucleic acid molecules set forth in SEQ ID NOS: 7 to 48 comprise one or more substitutions or modifications. In one embodiment, the nucleotides are substituted with locked nucleic acids (LNA).
1001611 In another preferred embodiment, the oligonucleotides target one or more regions of the nucleic acid molecules sense and/or antisense of coding and/or non-coding sequences IS associated with SIRTI and the sequences set forth as SEQ ID NOS: 1 to 6.
The oligonucleotides are also targeted to overlapping regions of SEQ ID NOS: I to 6.
1001621 Certain preferred oligonucleotides of this invention are chimeric oligonucleotides.
"Chimeric oligonucleotides" or "chimeras." in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA
hybrids.
By way of example, RNase H is a cellular endonuelease which cleaves the RNA
strand of an RNA :DNA duplex. Activation of RNitse II, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense modulation of gene expression.
Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used. compared to phosphorothioate deoxyoligonueleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessaty, associated nucleic acid hybridization techniques known in the art. In one preferred embodiment, a chimeric oligonticleotide comprises at least one region modified to increase target binding affinity, and, usually, a region that acts as a substrate for RNAse H. Affinity of an oligontieleotide for its target (in this case, a nucleic acid encoding nis) is routinely determined by measuring the Tilt of an oligonucleotideltarget pair, which is the temperature at which the oligonucleotide and target dissociate;
dissociation is detected spectrophotometrically. The higher the Tin, the greater is the affinity of the oligonucicotide for the target.
1001631 Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides. modified oligonucleotides, oligonucleosides and/or oligonucleotides mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, but arc not limited to, US
patent nos.
5,013,830; 5,149.797; 5, 220,007; 5,256.775; 5,366,878; 5.403311; 5,491,133;
5.565,350:
5.623,065: 5,652.355: 5,652,356; and 5,700.922.
1001641 In another preferred embodiment, the region of the oligonucicotide which is modified comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2.-Oalkyl. 2*-0-alkyl-0-alkyl or 2'-fluoro-modified nucleotide.
In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2' 0-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tai (i.e.. higher target binding affinity) than 2'-deoxyoligonucleotides against a given target. The effect of such increased affinity is to greatly enhance RNAi oligonucicotidc inhibition of gene expression. RNAsc H is a cellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target. and thus can greatly enhance the efficiency of RNAi inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis. In another preferred embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynueleotide. Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides.
A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. Oligonueleotides which contain at least one phosphorothioate modification are -35.
presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance.
Some desirable modifications can be found in De Mesmaeker et al. (1995) Ace. Chem. Res.,
28:366-374.
[00165] Specific examples of some preferred oligonucleotides envisioned for this invention include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2 --NH--0--CH2, CH,--N(CH3)--0--CH2 [known as a methylene(methylimino) or MMI backbone], CH2 --0--N (CH3)--CH2, CH2 ¨N (CH3)--N
(CH3)--CH2 and 0--N (CH3)--CH2 --CH2 backbones, wherein the native phosphodiester backbone is represented as 0--P--0--CH,). The amide backbones disclosed by De Mesmaeker et at. (1995) Acc. Chem. Res. 28:366-374 are also preferred. Also preferred are oligonucleotides having morpholino backbone structures (Summerton and Weller, U.S. Pat.
No. 5,034,506). In other preferred embodiments, such as the peptide nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al. (1991) Science 254, 1497). Oligonucleotides may also comprise one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 0(CH2)n CH3, 0(CH2)n NH2 or 0(CH2)n CH3 where n is from 1 to about 10; Cl to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3 ;
OCF3; 0--, S--, or N-alkyl; S--, or N-alkenyl; SOCH3; SO2 CH3; 0NO2; NO2; N3; NH2;
heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;
substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy [2'-0-CH2 CH2 OCH3, also known as 2'-0-(2-methoxyethyl)] (Martin et at., (1995) Hely. Chim. Acta, 78, 486). Other preferred modifications include 2'-methoxy (2'-0--CH3), 2'- propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
[00166] Oligonucleotides may also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleotides include nucleotides found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5- hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleotides, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5- bromouracil, hydroxymethyluracil, 8-azaguaninc, 7-dcazaguaninc, N6 (6-aminohcxyl)adcninc and 2,6-diaminopurine. (Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp75-77; Gebeyehu, G., (1987) etal. Nucl. Acids Res. 15:4513). A "universal"
base known in the art, e.g., inosine, may be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 C. (Sanghvi, Y. S., in Crooke, S. T.
and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.
276-278) and are presently preferred base substitutions.
[00167] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety (Letsinger et at., (1989) Proc.
Natl. Acad. Sci. USA 86, 6553), cholic acid (Manoharan et at. (1994) Bioorg.
Med. Chem. Let.
4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et at. (1992) Ann.
NY. Acad. Sci.
660, 306; Manoharan et at. (1993) Bioorg. Med. Chem. Let. 3, 2765), a thiocholesterol (Oberhauser et at., (1992) Nucl. Acids Res. 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et at. EMBO J. 1991, 10, 111; Kabanov et at. (1990) FEBS Lett. 259, 327; Svinarchuk et at. (1993) Biochimie 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero- 3-H-phosphonate (Manoharan etal. (1995) Tetrahedron Lett. 36, 3651; Shea etal.
(1990) Nucl.
Acids Res. 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et at. (1995) Nucleosides & Nucleotides, 14, 969), or adamantane acetic acid (Manoharan et al. (1995) Tetrahedron Lett. 36, 3651). Oligonucicotidcs comprising lipophilic moieties, and methods for preparing such oligonucleotides are known in the art, for example, U.S.
Pat. Nos.
5,138,045, 5,218,105 and 5,459,255.
[00168] It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleotide within an oligonucleotide.
The present invention also includes oligonucleotides which are chimeric oligonucleotides as hereinbefore defined.
[00169] In another embodiment, the nucleic acid molecule of the present invention is conjugated with another moiety including but not limited to abasic nucleotides, polyether, polyamine, polyamides, peptides, carbohydrates, lipid, or polyhydrocarbon compounds.
Those skilled in the art will recognize that these molecules can be linked to one or more of .. any nucleotides comprising the nucleic acid molecule at several positions on the sugar, base or phosphate group.
1001701 The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of one of ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling VA) to synthesize fluorescently labeled, biotinylated or other modified oligonucleotides such as cholesterol-modified oligonucleotides.
[00171] In accordance with the invention, use of modifications such as the use of LNA
monomers to enhance the potency, specificity and duration of action and broaden the routes of administration of oligonucleotides comprised of current chemistries such as MOE, ANA, FANA, PS etc (ref: Recent advances in the medical chemistry of antisense oligonucleotide by Uhlman, (2000) Current Opinions in Drug Discovety & Development Vol 3 No 2).
This can be achieved by substituting some of the monomers in the current oligonucleotides by LNA
monomers. The LNA modified oligonucleotide may have a size similar to the parent compound or may be larger or preferably smaller. It is preferred that such LNA-modified oligonucleotides contain less than about 70%, more preferably less than about 60%, most preferably less than about 50% LNA monomers and that their sizes arc between about 5 and 25 nucleotides, more preferably between about 12 and 20 nucleotides.
1001721 Preferred modified oligonueleotide backbones comprise, but not limited to, phosphorothioates. chiral phosphorothioates. phosphorodithioatcs.
phosphotriesters, aminoalkylphosphotriesters. methyl and other alkyl phosphonates comprising l'alkylene phosphonates and chiral phosphonatcs, phosphinates. phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramiclates, thionophosphoramidates.
thionoalkylphosphonates. thionoalkylphosphotriesters, and boranophosphates having nomial 3'-5' linkages. 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleotide units are linked 3*-5' to 5'-3' or 2'-S' to 5'-2'. Various salts, mixed salts and free acid forms arc also included.
1001731 .. Representative United States patents that teach the preparation of the above phosphorus containing linkages comprise, but arc not limited to, US patent nos. 3,687,808;
4,469,863; 4,476,301; 5,023.243; 5. 177,196: 5,188,897; 5.264.423: 5,276.019;
5,278,302;
5,286,717; 5,321,131; 5,399,676: 5,405,939: 5,453,496: 5,455, 233: 5,466,677;
5,476.925;
5.519,126; 5,536,821; 5.541,306; 5.550.111; 5,563, 253; 5,571,799; 5.587,361;
and 5,625.050.
1001741 Preferred modified oligonueleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part From the sugar portion of a nucleotide); siloxatte backbones; sulfide, sulfoxide and sulfone backbones;
folmacetyl and thiofonnacetyl backbones; methylene formacetyl and thioformacetyl backbones;
alkene containing backbones; stilt-mime backbones. methyleneimino and inethylenehydrazino backbones: sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and C1-12 component parts.
1001751 Representative United States patents that teach the preparation of' the above oligonucleosides comprise, but are not limited to. US patent nos. 5,034,506;
5,166.315:
5.185,444; 5,214,134; 5,216,141: 5,235.033: 5.264, 562; 5, 264,564; 5,405.938;
5,434.257;
5,466,677; 5,470,967; 5,489,677; 5,541.307; 5,561,225; 5,596, 086; 5.602.240;
5,610,289;
5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5.663.312: 5.633,360;
5,677,437;
and 5,677,439.
1001761 In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an olig,onticleolide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are retained and are bound .. directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
Representative United States patents that teach the preparation of PNA
compounds comprise, but are not limited to, US patent nos. 5,539,082: 5,714,331: and 5,719,262.
Further teaching of PNA compounds can be found in o Nielsen et at.. Science, 1991, 254. 1497-1500.
1091771 In another preferred embodiment of the invention the oligonucleoticles with phosphorothioate backbones and oligonucicosides with heteroatom backbones, and in particular- C1-12-NH-O-CH2-,-CH2-N (C1-13)-0-C1-12-known us a methylene (methylimino) or MM1 backbone.- CH2-0-N (CH3)-CH2-.-CH2N(CH3)-N(CH3) CH2-and-O-N(CH3)-CH2-CH2- wherein the native phosphodiester backbone is represented as-O-P-0-0-12-of the above referenced US patent no. 5,489.677. and the amide backbones of the above referenced US patent no. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced US patent no. 5.034,506.
101781 Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; I':
0-. S-, or N-alkyl: 0-. S-. or N-alkenyl: 0-, S-or N-alkynyl: or 0 alkyl-0-alkyl. wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C to CO alkyl or C2 to CO
alkenyl and alkynyl. Particularly preferred are 0 (CI12)n OinC113. 0(012)n,OCI-13, 0(a-12)11Na-12. 0(012)nC113, 0(('112.)nONH2. and 0(CH2nON(CH2)ncH3)2 where n and m can be from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C to CO. (lower alkyl, substituted lower alkyl, alkaryl.
aralkyl, 0-alkaryl or 0-aralkyl, SH, SCH3. OCN. Cl, Br. CN, CF3, OCF3, SOCI13. SO2C1-13. 0NO2. NO2, N3, NH2, heterocycloalkyl, hetcrocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl. an RNA cleaving group, a reporter group. an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification comprises 2'-methoxyethoxy (2'-0-CH2CF1200-13. also known as 2'4)-(2- methoxyethyl) or 2'-M0E) (Martin et al., (1995) Halt'. Chun.
Acta, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification comprises 2'-dimethylaminooxyethoxy, i.e. , a 0(CH2)20N(C1-13)2 group, also known as 2'-DMA0E, as -40.
described in examples herein below, and 2'- climethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or DMAEOE:), i.e.. 2'-0-0-12-0-CH2-N
(CH2)2.
1001791 Other preferred modifications comprise 2'-methoxy (2'-0 CH3), 2'-aminopropoxy (2'-O CI 12C1-12042N112) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar miluctics such as cyclobutyl moieties in place of the pentofuntnosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures comprise, but are not limited to, US patent nos.
4,981.957: 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5.446,137; 5,466,786;
5.514, 785:
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873;
5,616, 265; 5,658,873; 5.670.633: and 5,700,920.
1001801 Oligonucleotides may also comprise nucleobase (often referred to in the art simply IS as "base") modifications or substitutions. As used herein, "unmodified"
or "natural"
nucleotides comprise the purine bases adenine (A) and guanine (G), and the pyrimidinc bases thymine (T), cytosine (C) and uracil (U). Modified nucleotides comprise other synthetic and natural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthinc, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine. 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo mei!, cytosine and thymine, 5-uracil (pseudo-uracil 4-thiouracil, 8-halo. 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-tritluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladeninc, 8-azaguanine and 8-azattdeninc, 7-dcazaguanine and 7-dcazatidenine and 3-dcazaguaninc and 3-dcazaadenine.
1001811 Further, nucleotides commise those disclosed in United States Patent No.
3.687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science And Engineering', pages 858-859, Kroschwitz, J.1., ed. John Wiley & Sons, 1990, those disclosed by Englisch et at,'Angewandle Chemie, International Edition', 1991, 30, page 613. and those disclosed by Sanghvi, Y.S.. Chapter 15. 'Antisense Research and Applications', pages 289-302, Crooke, S.T. and Lebleu. B. ca., CRC Press, 1993. Certain of these nucleotides are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These comprise 5-substituted pyrimidines, 6- azapyrirnidines and N-2. N-6 and 0-6 substituted purifies. comprising 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosinc. 5-methyleytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2cC (Sanahvi, V.S.. ('rooke, S.T. and Leblett, B., eds, 'Antisense Research and Applications', CRC Press, Boca Raton. 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-Omethoxyethyl sugar modifications.
1001821 Representative United States patents that teach the preparation of the above noted modified nucleotides as well as other modified nucleotides comprise, but are not limited to, US patent nos. 3,687.808, as well as 4,845.205; 5,130.302; 5,134,066; 5.175, 273; 5. 367,066;
5,431272: 5,457,187; 5.459.255; 5.484,908; 5,502,177; 5.525.711; 5,552.540;
5,587,469;
5.596.091; 5.614,617: 5,750,692, and 5,681,941.
1001831 Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotidc one or more moieties or conjugates, which enhance the activity.
cellular disnibution, or cellular uptake of the oligonucicotide.
1001841 Such moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et aL, Proc. Natl. .4cad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.. (1994) MmHg. Med. Client. Let., 4. 1053-1060). a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.. (1992) .4nn. N. )'. Acad. Sci.. 660, 306-309;
Manoharan et al..
(1993) Biooty. Med. ('hem. Let.. 3, 2765-2770). a thioeholesierol (Oberhauser ci at. (1992) ...his RCA., 20, 533-538), an aliphatic chain. e.g., dodecandiol or undecyl residues (Kabanov A. (1990) FESS Lett., 259, 327-330: Svinarchuk etal., (1993) Bioc.hiinie 75, 49-54), a phospholipid, e.g., di-hexadecyl-rue-glyeerol or triethylammonium 1.2-di-O-ltexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., (1995) Tetrahedron Len.. 36, 3651-3654;
Shea et al.. (1990) Nucl. Acids Res.. 18. 3777-3783). a polyamine or a polyethylene glycol chain (Maneharan er iiI.. (1995) Nucleosides & Nucleotides, 14, 969-973). or adamantane acetic acid (Manohartin et al.. (1995) Tetrahedron Lett., 36, 3651-3654), a palmityl moiety (Mishrti et al., (1995) Siochint. Biophys. Acta, 1264, 229-237), or an octadecylamine or hexylarnino-carbonyl-t oxycholesterol moiety (Crooke et al., (1996) J.
Pharmacrit rho... 277, 923-937).
1001851 Representative United States patents that teach the preparation of such oligonucleotides conjugates comprise, but are not limited to. US patent nos.
4,828,979;
4,948.882; 5,218,105; 5,525,465; 5.541,313; 5,545.730: 5,552. 538; 5.578,717.
5,580,731;
5,580.731; 5,591,584; 5,109,124; 5,118,802, 5,138,045; 5.414.077: 5.486, 603;
5,512,439;
5.578,718; 5,608,046; 4.587,044; 4,605,735; 4.667,025; 4,762, 779; 4.789,737;
4,824,941:
4.835.263; 4,876,335; 4.904,582; 4,958,013; 5,082, 830; 5,112.963: 5.214,136;
5,082,830;
5,112.963; 5.214,136; 5. 245.022: 5,254.469: 5.258,506; 5,262,536: 5.272,250;
5,292,873;
5,317.098; 5.371,241, 5,391, 723: 5,416.203, 5,451,463; 5,510,475: 5.512.667;
5,514.785; 5, 565,552; 5,567,810; 5,574,142; 5,585,481; 5.587.371; 5.595,726: 5,597,696;
5.599,923;
5,599, 928 and 5,688,941.
1001861 DIV; diseoveiy: The compounds of the present invention can also be applied in the areas of drug discovery and target validation. Tie present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between Sirtuin 1 (SIRT1) polynucleotides and a disease state, phenotype, or condition. These methods include detecting or modulating Sirtuin (SIRT1) polynucleotides comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of Sirtuin (SIRT I ) polynucleotides and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.
Assessing Up-regulation or Inhibition of Gene Expression:
1001871 Transfer of an exogenous nucleic acid into a host cell or organism can be assessed by directly detecting the presence of the nucleic acid in the cell or organism. Such detection can be achieved by several methods well known in the art. For example, the presence of the exogenous nucleic acid can be detected by Southern blot or by a polymerase chain reaction (PCR) technique using primers that specifically amplify nucleotide sequences associated with the nucleic acid. Expression of the exogenous nucleic acids can also be measured using conventional methods including gene expression analysis. For instance, mRNA
produced from an exogenous nucleic acid can be detected and quantified using a Northern blot and reverse transcription PCR (RT-PCR).
1001881 Expression of RNA from the exogenous nucleic acid can also be detected by measuring an enzymatic activity or a reporter protein activity. For example, antisense modulatory activity can be measured indirectly as a decrease or increase in target nucleic acid expression as an indication that the exogenous nucleic acid is producing the effector RNA.
Based on sequence conservation, primers can be designed and used to amplify coding regions of the target genes. Initially, the most highly expressed coding region from each gene can be used to build a model control gene, although any coding or non coding region can be used.
Each control gene is assembled by inserting each coding region between a reporter coding region and its poly(A) signal. These plasmids would produce an mRNA with a reporter gene in the upstream portion of the gene and a potential RNAi target in the 3' non-coding region.
The effectiveness of individual antisense oligonucleotides would be assayed by modulation of the reporter gene. Reporter genes useful in the methods of the present invention include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), antibiotic resistance determination.
Kits, Research Reagents, Diagnostics, and Therapeutics [00189] The compounds of the present invention can be utilized for diagnostics, therapeutics, and prophylaxis, and as research reagents and components of kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
[00190] For use in kits and diagnostics and in various biological systems, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, are useful as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.
[00191] As used herein the term "biological system" or "system" is defined as any organism, cell, cell culture or tissue that expresses, or is made competent to express products of the Sirtuin 1 (SIRT1) genes. These include, but are not limited to, humans, transgenic animals, cells, cell cultures, tissues, xenografts, transplants and combinations thereof.
[00192] As one non limiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, .. cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds that affect expression patterns.
[00193] Examples of methods of gene expression analysis known in the art include DNA
arrays or microarrays (Brazma and Vilo, (2000) FEBS Lett., 480, 17-24; Celis, et al., (2000) FEBS Lett., 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., (2000) Drug Discov. Today, 5, 415- 425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, (1999) Methods Enzytnol., 303, 258-72), TOGA
(total gene expression analysis) (Sutcliffe, et al., (2000) Proc. Natl. Acad. Sci. U.S.A., 97, 1976-81), protein arrays and proteomics (Celis, et al., (2000) FEBS Lett., 480, 2-16;
Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., (2000) Anal. Biochem. 286, 91-98;
Larson, et al., (2000) Cytometty 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, (2000) Cum Opin. Microbiol. 3, 316-21), comparative genomic hybridization (Carulli, et al., (1998) J. Cell Biochent. Suppl., 31, 286-96), FISH
(fluorescent in situ hybridization) techniques (Going and Gusterson, (1999) Eur. J. Cancer, 35, 1895-904) and mass spectrometry methods (To, Comb. (2000) Chem. High Throughput Screen, 3, 235-41).
[00194] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Sirtuin 1 (SIRT1). For example, oligonucleotides that hybridize with such efficiency and under such conditions as disclosed herein as to be effective Sirtuin 1 (SIRT1) modulators are effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding Sirtuin 1 (SIRT1) and in the amplification of said nucleic acid molecules for detection or for use in further studies of Sirtuin 1 (SIRT1). Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding Sirtuin 1 (SIRT1) can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabeling of the oligonueleotide, or any other suitable detection means. Kits using such detection means for detecting the level of Sirtuin 1 (SIRT1) in a sample may also be prepared.
[00195] The specificity and sensitivity of antisense are also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
[00196] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Sirtuin 1 (SIRT1) polynucleotides is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of Sirtuin 1 (SIRT1) modulator. The Sirtuin 1 (SIRT1) modulators of the present invention effectively modulate the activity of the Sirtuin 1 (SIRT1) protein or modulate the expression of the Sirtuin 1 (SIRT1) protein. In one embodiment, the activity or expression of Sirtuin 1 (SIRT1) in an animal is inhibited by about 10% as compared to a control. Preferably, the activity or expression of Sirtuin 1 (SIRT1) in an animal is inhibited by about 30%. More preferably, the activity or expression of Sirtuin 1 (SIRT1) in an animal is inhibited by 50%
or more. Thus, the oligomeric compounds modulate expression of Sirtuin 1 (SIRT1) mRNA by at least 10%, by at least 50%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100% as compared to a control.
[00197] In one embodiment, the activity or expression of Sirtuin 1 (SIRT1) and/or in an animal is increased by about 10% as compared to a control. Preferably, the activity or expression of Sirtuin 1 (SIRT1) in an animal is increased by about 30%. More preferably, the activity or expression of Sirtuin 1 (SIRT1) in an animal is increased by 50%
or more. Thus, the oligomeric compounds modulate expression of Sirtuin 1 (SIRT1) mRNA by at least 10%, by at least 50%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100% as compared to a control.
[00198] For example, the reduction of the expression of Sirtuin 1 (SIRT1) may be measured in serum, blood, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids. tissues or organs being analyzed contain a nucleic acid molecule encoding Sirtuin 1 (SIRT1) peptides and/or the Sirtuin 1 (S I RT I ) protein itself.
1001991 The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.
Conjugates 1002001 Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleolide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols. polyethers, groups that enhance the phamtacodynamic properties of limners, and groups that enhance the pharmacokinetic properties of oligomers. Typicalconjugatc groups include cholesterols, lipids, phospholipids.
biotin, phenazine, folate, phenanthridine. anthraquinone, acridine, fluorescents, rhodamines, coumarins. and dyes. Groups that enhance the pharmacodynamie properties, in the context of this invention, include groups that improve uptake. enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
Groups that enhance the pharmacokinetic properties. in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention.
Representative conjugate groups arc disclosed in International Patent Application No.
PC'T!US92109196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,1(60.
Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioethcr, e.g., hexy1-5- tritylthiol. a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glyeero-3-Flphosphonate. a polyamine or a polyethylene glycol chain, or adamantanc acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example.
aspirin, warfarin, phenylbutazone. ibuprofen. suprofen. fenbufen, ketoprolen, (S)-(+)-pranoprofen, carpro fen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
1002011 Representative United States patents that teach the preparation of such oligonucicotides conjugates include, but arc not limited to. U.S. Pat. Nos.
4.828,979:
$ 4.948,882: 5,218,105; 5,525,465; 5,541,313; 5,545.730; 5,552,538;
5,578.717, 5,580,731:
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;
5,512,439;
5.578,718; 5,608,046; 4,587,044; 4,605.735; 4,667.025; 4,762.779; 4,789,737:
4,824,941:
4.835,263; 4,876,335; 4,904,582; 4,958,013; 5,082.830; 5,112,963; 5,214,136;
5.082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250:
5.292.873;
5,317.098: 5,371,241, 5,391,723; 5,416,203, 5.451,463; 5,510,475; 5,512.667;
5,514,785;
5,565.552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5.595,726; 5,597,696:
5.599.923;
5,599.928 and 5,688.941.
FormulaiionA
1002021 The compounds of the invention may also be admixed. encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as lot-example, liposomes, receptor-targeted molecules. oral, rectal, topical or other formulations, for assisting in uptake. distribution andior absorption.
Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos.
5,108.921; 5.354,844:
5.416,016: 5,459,127; 5.521,291; 5,543,165: 5.547,932; 5,583.020; 5,591,721:
4.426,330;
4,534,899: 5,013,556; 5.108,921; 5,213,804; 5,227,170; 5.264.221; 5,356,633;
5,395,619;
5,416,016; 5,417,978; 5,462,854: 5,469,854; 5,512,295: 5.527,528; 5.534,259:
5,543,152:
5,556.948; 5,580,575; and 5,595,756.
1002031 Although, the antisense oligonucleotides do not need to be administered in the context of a vector in order to modulate a target expression and/or function, embodiments of the invention relates to expression vector constructs for the expression of antisense olig,onueleotides, comprising promoters, hybrid promoter gene sequences and possess a strong constitutive promoter activity, or a promoter activity which can be induced in the desired case.
1002941 In an embodiment. invention practice involves administering at least one of the foregoing antisense oligonucleotides with a suitable nucleic acid delivery system. In one embodiment, that system includes a non-viral vector operably linked to the polynucleotide.
Examples of' such nonviral vectors include the oligonucleotidc alone (e.g. any one or more of SEQ ID NOS: 7 to 48 or in combination with a suitable protein, polysaccharide or lipid formulation.
1002051 Additionally suitable nucleic acid delivery systems include viral vector, typically sequence from at least one of an adenovirus, adenovirus-associated virus (AAV), helper-.5 dependent adenovirus, retrovirus, or hcmagglutinatin virus of Japan-liposome (HVJ) complex.
Preferably, the viral vector comprises a strong cukaryotic promoter operably linked to the polynucleotide e.g.. a cytomegalovirus (CMV) promoter.
1002061 Additionally preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney nutrine leukemia viruses and WV-based viruses One preferred HIV-based viral vector comprises at least two vectors wherein the gag and poi genes are from an HIV genomc and the env gene is from another virus.
DNA viral vectors arc preferred. These vectors include pox vectors such as orthopox or avipos vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector [Geller.
A.1. et al., (1995) J. Neurochent, 64: 487; Lim. F., etal., in DNA Cloning: Mammalian Systems, D.
Glover, Ed.
(Oxford Univ. Press. Oxford England) (1995); Geller, A.I. et al.,(1993) Proc Natl. Acad. Sc!.:
U.S.A.:90 7603: Geller, Al., etal.. (1990) Proc Natl. Acta Sc! USA: 87:11491, Adcnovirus Vectors [LeGal LaSalle et al.. Science, 259:988 (1993); Davidson. et al., (1993) A'at. Genet.
3: 219; Yang, etal.. (1995).!. Vim!. 69: 20041 and Adeno-associated Virus Vectors [Kapliit, M.G.. et a/.. (1994 ) Nat. Genet. 8:148].
1002071 The antisensc compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
1002081 The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.c., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonuelcotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Put. No. 6.287.860.
1002091 The present invention also includes pharmaceutical compositions and formulations that include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'-0-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful.
[00210] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
[00211] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
[00212] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.
[00213] Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 lam in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug that may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase.
Mieroemulsions are included as an embodiment of the present invention.
Emulsions and their uses are well known in the art and are further described in U.S. Pat. No.
6,287,860.
1002141 Formulations of the present invention include liposornal formulations. As used in the present invention, the term "liposorne" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes arc unilamellar or multilamellar vesicles which have a membrane formed From a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged arc believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
1002151 Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids. When incorporated into liposomcs, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomeslacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860.
1002161 The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products.
formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S.
Pat. No. 6,287.860.
1002171 In one embodiment. the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also .. enhance the pertneability of lipophilic drugs. Penetration enhancers may be cla.ssified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chclating nonsurfitctants. Penetration enhancers and their uses arc further described in U.S. Pat. No. 6,287,860.
1002181 One of skill in the art will recognize that formulations are routinely designed according to their intended use. i.e. route of administration.
1002191 Preferred formulations for topical administration include those in which the olionucleotides of the invention are in admixture with a topical delivery agent such as lipids.
liposomes, fatty acids, fatty acid esters, steroids, dictating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoyl-phosphatidyl DOPE
ethanolamine, dimyristoylphosphatidyl cholinc DMPC, distearolyphosphatidyl choline) negative (e.g.
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.
dioleoyltetrarnethylaminopropyl DOTAP and dioleoyl-phosphatidyl ethanolamine DOTMA).
1002201 For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters. pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860.
1002211 Compositions and formulations for oral administration include powders or granules, microparticulaies, nanoparticulates. suspensions or solutions in water or non-aqueous media. capsules, gel capsules. sachets, tablets or minitableis.
Thickeners, flavoring agents, diluents, emulsifiers. dispersing aids or binders may be desirable.
Preferred oral formulations are those in which oligonucleotides of' the invention are administered in conjunction with one or more penetration enhancers surfactants and dictators.
Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
Preferred bile acids/salts and fatty acids and their uses arc further described in U.S. Pat. No.
6,287,860. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids!salts. A
particularly preferred combination is the sodium salt of Laurie acid, capric acid and UDCA.
Further penetration enhancers include polyoxyethylene-9-lauryl ether.
polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles. or complexed to form micro or namoparticles.
Oligonucicotide complexing agents and their uses are further described in U.S.
Pat. No.
6,287.860.
1002221 Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to. penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
1002231 Certain embodiments of the invention provide pharmaceutical compositions containing one or more olicomeric compounds and one or more other chemotherapeutic agents that function by a non-antisense mechanism. Examples of' such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as datmorubiein.
datmomycin, dactinomycin, dosortibicin, epirubicin, idarubicin. esorubicin.
bleomycin.
mafosfitmide, ifosfitmide. cytosine arabinoside, bischloroethyl- nitrosurea, busulfitn.
mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention.
Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
[00224] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. For example, the first target may be a particular antisense sequence of Sirtuin 1 (SIRT1), and the second target may be a region from another nucleotide sequence. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same Sirtuin 1 (SIRT1) nucleic acid target. Numerous examples of antisense compounds are illustrated herein and others may be selected from among suitable compounds known in the art. Two or more combined compounds may be used together or sequentially.
Dosing:
[00225] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 pig to 100 g per kg of body weight, and may be given once or more daily. weekly. monthly or yearly, or even once every 2 to 20 years.
Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 lig to 100 g per kg of body weight. once or more daily. to once every years.
1002261 While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with 15 the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments.
By their citation of various references in this document. Applicants do not admit any particular reference is "prior art" to their invention.
Embodiments of inventive compositions and methods are illustrated in the following examples.
EXAMPLES
25 1002281 The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in pmportions and alternatives in elements of the components shown will be apparent to those skilled in the art and arc within the scope of embodiments of the present invention.
Evanple I: Design p/. antisense oligonucleothks specific ftir a nucleic acid puilecule 30 antisense anchor sense strand qrSirtuin I (S I RT1 ) polynticleolide 1002291 As indicated above the term "oligonucleotide specific for" or "oligonucleotide targets" refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene.
[00230] Selection of appropriate oligonucleotides is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products.
Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity. These procedures allow the selection of oligoncueltodes that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species. One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.
[00231] An antisense compound is "specifically hybridizable" when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays [00232] The hybridization properties of the oligonucleotides described herein can be determined by one or more in vitro assays as known in the art. For example, the properties of the oligonucleotides described herein can be obtained by determination of binding strength between the target natural antisense and a potential drug molecules using melting curve assay.
[00233] The binding strength between the target natural antisense and a potential drug molecule (Molecule) can be estimated using any of the established methods of measuring the strength of intermolecular interactions, for example, a melting curve assay.
[00234] Melting curve assay determines the temperature at which a rapid transition from double-stranded to single-stranded conformation occurs for the natural antisense/Molecule complex. This temperature is widely accepted as a reliable measure of the interaction strength between the two molecules.
1002351 A melting curve assay can be performed using a eDNA copy of the actual natural antisense RNA molecule or a synthetic DNA or RNA nucleotide corresponding to the binding site of the Molecule. Multiple kits containing all necessary reagents to perform this assay arc TM
available (e.g. Applied Biosystems Inc. MeltDoctor kit). These kits include a suitable buffer solution containing one of the double strand DNA (dsDNA) binding dyes (such as ABI FIRM
dyes, SYBR Green, SYTO, etc.). The properties of the dsDNA dyes are such that they emit almost no fluorescence in free form, but are highly fluorescent when bound to dsDNA.
1002361 To perform the assay the cDNA or a corresponding oligonucleotide are mixed with Molecule in concentrations defined by the particular manufacturer's protocols.
The mixture is heated to 95 C to dissociate all pre-formed dsDNA complexes. then slowly cooled to room temperature or other lower temperature defined by the kit manufacturer to allow the DNA
molecules to anneal. The newly formed complexes arc then slowly heated to 95 C
with simultaneous continuous collection of data on the amount of fluorescence that is produced by the reaction. The fluorescence intensity is inversely proportional to the amounts of dsDNA
present in the reaction. The data can be collected using a real time PCR
instrument compatible TM TM
with the kit (e.g.AB1's StepOne Plus Real Time PCR System or LightTyper instrument.
Roche Diagnostics, Lewes, UK).
1002371 Melting peaks arc constructed by plotting the negative derivative of fluorescence with respect to temperature (-d(Fluorescence)idT) on the y-axis) against temperature (x-axis) TM
using appropriate software (for example LighiTyper (Roche) or SDS Dissociation Curve.
ABI). The data is analyzed to identify the temperature of the rapid transition from dsDNA
complex to single strand molecules. This temperature is called Tm and is directly proportional to the strength or intentction between the two molecules. Typically. Tin will exceed 40 'C'.
Example 2: Modulation ql mlynuckatide3 Materials and Methods:
Treatment M'IlepG2 cells will? naked amisense oligonueleotides:
1002381 I1ep02 cells from ATCC (cat# HB-8065) were grown in growth media (MENVEBSS (1-lyclone cat ;I-SI-130024. or Mediatech cat # MT-10-010-CV) +10%
1:13S
(Mediatech catO MT35- 011-CV) + penicillinistreptomycin (Mediatech cat# MT30-002-CI)) at 37cC and 5% CO2. One day before the experiment the cells were replated at the density of 0.5 x 1 Osiml into 6 well plates and incubated at 37 C and 5% CO2. On the day of the experiment the media in the 6 well plates was replaced with 1.5 mitwell of fresh growth media. All antisense oligonucleotides were diluted in water to the concentration of 20 pM. 2 pl of this solution was incubated with 400 pl of Opti-MEM media (Gibco cat#31985-070) and TM
4 ul of Lipofcctamine 2000 (Invitroaen cart/ 11668019) at room temperature for 20 min and applied to each well of the 6 well plates with HcpG2 cells. Similar mixture including 2 IA of water instead of the oligonucleotide solution was used for the mock-transfected controls.
After 3-18 h of incubation at 37 C and 5% CO2 the media was changed to fresh growth media. 72 It after addition of antisense oligonucleotides the cells were redosed as described in above. 48h after the second addition of antisense oligonucleotides the media was removed and RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat #
TM
Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cats 74181) Following the manufacturers' instructions. 600 lig of RNA was added to the reverse transcription reaction performed using Verso cDNA kit from Thermo Scientific (ctit#AB1453B) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to TM
monitor gene expression by real time PCR using ABI 'ragman Gene Expression Mix TM
(cat#4369510) and primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00202021...m1 by Applied Biosystems Inc.. Foster City CA).
The following PCR cycle was used: 50 C' for 2 min. 95 C for 10 min, 40 cycles of (95 C for 15 seconds, 60 C for I min) using StcpOne Plus Real Time PCR Machine (Applied Biosystems).
Fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in 'SS-normalized dCt values between treated and mock-transfectcd samples.
Results:
1002391 Real time PCR results show that the levels of the SIRTI mRNA in HepG2 cells significantly increased 48 h after treatment with some antisense oligonucleotides to SIRTI
antisense CV396200 (Figure 2. 3A).
1002401 Real Time PCR results show that levels of SIRTI mRNA in 1lepG2 cells are significantly increased in one of the oligonucleotides designed to SIRTI
antisense CV396200.
The burs denoted us CUR- 1230. CUR- 123 1, CUR- 1 232 and CUR-1230 correspond to SEQ
ID NOs:33-36. (Fig 6) 1002411 Real Time PCR results show that levels of SIRTI mRNA in HepG2 cells are significantly increased in two of the oligonucleotides designed to SIRTI
antisense CV428275.
The bars denoted as CUR-1302, CUR-1304, CUR- 1303 and CUR-I305 correspond to SEQ
ID NOs:37-40. (Fig 7) 1002421 The results show that a significant increase in SIRTI mRNA levels in HepG2 cells 48 hours after treatment with one of the oligonucleotides designed to SIRT
antisense BE7I7453. The burs denoted as CUR-I264, CUR1265 and CUR-1266 correspond to SEQ
ID
NOs.: 41-43 respectively. (Fig 8) 1002431 The results show that the levels of the SIRT1 mRNA in FlepG2 cells are significantly increased 48 h after treatment with three of the oligonucicotides designed to sum antisense AV7I 8812. The bars denoted as CUR-I294, CUR-1297, CUR-1295, CUR-1296 and CUR-I298 correspond to SEQ ID NOs.: 44-48 respectively. (Fig 9) Treatment qf Vero76 cells with antisense oligonuclemides:
1002441 Vcro76 cells from ATCC (cat# CRL-1587) were grown in growth media (MEM'EBSS (Hyclone cat #SH30024. or Nlediatech cat # MT-10-010-CV) -10% FBS
(Mediated) cat# 011-CV)+ penicillin/streptomycin (Mediatech cut;! W30-0024.1)) at 37 C and 5% CO2. One day before the experiment the cells were replated at the density of 1.5 105/m1 into 6 well plates and incubated at 37 C and 5% CO2. On the day of the experiment the media in the 6 well plates was changed to fresh growth media. All antisense oligonucicotides were diluted in water to the concentration of 20 1.1114. 2 pl of this solution was incubated with 400 III of Opti-MEM media (Gibco cat431985-070) and 4 ul of Lipofcetamine 2000 (Invitrogen cat# 11668019) at room temperature for 20 min and applied to each well of the 6 well plates with Ven)76 cells. Similar mixture including 2 Id of water instead of the oligonucleotide solution was used for the mock- transfected controls. After 3-18 h of incubation at 37 C and 5% CO2 the media was changed to fresh growth media. 48 h after addition of antisense oligonucleotidcs the media was removed and RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat # 2.3105) or RNeasy Total RNA Isolation kit from Qiagcn (cat# 74181), following the manufacturers instructions. 600 ng of RNA was added to the reverse transcription reaction performed using Verso cDNA kit from Themo Scientific (cat#AB1453B) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PCR using A131 Taqmun Gene EXIMS:ii011 Mix (cab14369510) and primers/probes designed by ABI (Applied Biosystems Tagman Gene Expression Assay:
Hs00202021_ml by Applied Biosystems Inc.. Foster City CA). The following PCR cycle was used: 50 C for 2 9.5 C for 10 mitt. 40 cycles of (95 C for IS seconds. 60 C for I min) using StepOne Plus Real Time PCR Machine (Applied Biosystems). Fold change in gene expression after treatment with antisensc oligonucleolides was calculated based on the difference in I8S-normalized dCt values between treated and mock-transfected samples.
Results:
[00245] Real time PCR results show that the levels of the SIRT1 mRNA in Vero cells significantly increased 48 h after treatment with antisense oligonucleotides to SIRT1 antisense CV396200 (Figure 3B).
Example 3: Modulation of SIR Ti Gene Expression Materials and Methods Treatment of HepG2 cells with naked antisense oligonucleotides:
[00246] HepG2 cells from ATCC (cat# HB-8065) were grown in growth media (MEM/EBSS (Hyclone cat #SH30024, or Mediatech cat # MT-10-010-CV) +10% FBS
(Mediatech cat# MT35- 011-CV) + penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37 C and 5% CO2. One day before the experiment the cells were replated at the density of 0.5 x 105/m1 into 6 well plates and incubated at 37 C and 5% CO2. On the day of the experiment the media in the 6 well plates was replaced with 1.5 ml/well of fresh growth media. All antisense oligonucleotides were diluted in water to the concentration of 20 [tM. 2 111 of this solution was incubated with 400 pi of Opti-MEM media (Gibco cat#31985-070) and 4 ul of Lipofectamine 2000 (Invitrogen cat# 11668019) at room temperature for 20 min and applied to each well of the 6 well plates with HepG2 cells. Similar mixture including 2 iõt1 of water instead of the oligonucleotide solution was used for the mock-transfected controls.
After 3-18 h of incubation at 37 C and 5% CO2 the media was changed to fresh growth media. 72 h after addition of antisense oligonucleotides the cells were redosed as described in above. 48h after the second dosing of antisense oligonucleotides the media was removed and RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat #
Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat# 74181) following the manufacturers' instructions. 600 ng of RNA was added to the reverse transcription reaction .. performed using Verso cDNA kit from Thermo Scientific (cat#AB1453B) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PCR using AB1 Taqman Gene Expression Mix (cat#4369510) and primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00202021_m 1 by Applied Biosystems Inc., Foster City CA).
The following PCR cycle was used: 50 C for 2 min, 95 C for 10 min, 40 cycles of (95 C for 15 seconds, 60 C for 1 min) using StepOne Plus Real Time PCR Machine (Applied Biosystems).
Fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in 18S-normalized dCt values between treated and mock-transfected samples.
[002471 Primers and probe for the custom designed Taqman assay for exon 4:
AACTGGAGCTGGGGTGTCTGTTTCA (SEQ ID NO:49) of the SIRT1 natural antisense CV396200.
Forward Primer Seq. CCATCAGACGACATCCCTTAACAAA (SEQ ID NO: 50) Reverse Primer Seq. ACATTATATCATAGCTCCTAAAGGAGATGCA (SEQ ID NO: 51) Reporter Seq. CAGAGTTTCAATTCCC (SEQ ID NO: 52) Results:
1002481 The results show that the levels of the SIRT1 mRNA in HepG2 cells are significantly increased 48 h after treatment with one of the siRNAs designed to sirtas (sirtas_5, P=0.01). In the same samples the levels of sirtas RNA were significantly decreased after treatment with sirtas 5, but unchanged after treatment with sirtas_6 and sirtas 7, which also had no effect on the SIRT1 mRNA levels (Fig. 1B). sirtas 5, sirtas_6 and sirtas_7 correspond to SEQ ID NO: 30, 31 and 32 respectively.
Treatment of primary monkey hepatoeytes.
1002491 Primary monkey hepatocytes were introduced into culture by RxGen Inc.
and plated in 6 well plates. They were treated with oligonucleotides as follows.
The media in the 6 well plates was changed to fresh growth media consisting of William's Medium E
(Sigma cat#W4128) supplemented with 5% 113S, 50 U/ml penicillin and 50 ug/m1 streptomycin, 4 ug/ml insulin, 1 uM dexamethasone, 10 ug/ml Fungin (InVivogen, San Diego CA).
All antisense oligonucleotides were diluted to the concentration of 20 p.M. 2 [d of this solution was incubated with 400 il of Opti-MEM media (Gibco cat#31985-070) and 4 pl of Lipofectamine 2000 (Invitrogen cat# 11668019) at room temperature for 20 min and applied to each well of the 6 well plates with cells. Similar mixture including 2 111 of water instead of the oligonucleotide solution was used for the mock-transfected controls. After 3-18 h of incubation at 37 C and 5% CO2 the media was changed to fresh growth media. 48 h after addition of antisense oligonucleotides the media was removed and RNA was extracted from the cells using S V Total RNA Isolation System from Promega (cat # Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat# 74181) following the manufacturers' instructions. 600 ng of RNA was added to the reverse transcription reaction performed using Verso cDNA kit from Thermo Scientific (cat#AB1453B) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PCR using ABI Taqman Gene Expression Mix (cat#4369510) and primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay:
Hs00978340_ml by Applied Biosystems Inc., Foster City CA). The following PCR cycle was used: 50 C for 2 min, 95 C for 10 min, 40 cycles of (95 C for 15 seconds, 60 C for I min) using Mx4000 thermal cycler (Stratagem). Fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in NS-normalized en values between treated and mock-transfectcd samples.
Results:
1002501 The results are shown in Figure. 5. Real time PCR results show an increase in SIRTI mRNA levels after treatment with an oligonueleotide against S1RT I
antisense.
to Example 4: Efficacy and Duration 44ction Study ocCUR 96$ in the African Green llonkey 1002511 The objective of this study was to assess and compare the effect of antisense knockdown of the discordant noncoding antisense sequences that regulate the SIRT I genes following intravenous administration in a nonhuman primate model. The antisense oligonucicotide test articles designed to inhibit the SIRTI regulatory sequences were designated as CUR 963.
CUR 963: +G* T*C*T*G*A*T*G*G*-FA*.t.G*+A (SEQ ID N(); 26).
CUR 962 (control): 4-G*+C*T*A*G*T*C*T*G++T*-rf*-1-G (SEQ ID NO:53).
REGULATORY TEST GUIDELINES
1002521 This study was designed in accordance with accepted toxicological principles and to comply with International Conference of Harmonization (ICH) Harmonized Tripartite Guidelines (Non-Clinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals I('H M3(m), 2000 November 9), and generally accepted procedures for the testing of therapeutic agents.
TEST AND CONTROL ARTICLES
Test Article Identity and Preparation 1002531 The test article. CUR-963, is a chemically stabilized antisense oligonucleotide.
The vehicle for intravenous delivery is phosphate-buffered saline (PBS).
Vehicle characterization 4)1-[00254] For the PBS vehicle, the composition, batch number, expiry date and storage conditions (temperature and light/dark) was obtained from the supplier.
Test Article Storage and Handling [00255] The test substance and vehicle were stored according to the received storage conditions supplied by the Sponsor and manufacturer, accordingly.
Analysis of the Test Article Formulations [00256] Samples of the test article formulation will be cryopreserved for analysis of the concentration, stability and homogeneity of the test substance formulations.
TEST SYSTEM RATIONALE
[00257] The primate is a suitable non rodent species, acceptable to regulatory authorities as an indicator of potential hazards, and for which extensive background data are available. The African green monkey specifically is a highly clinically relevant model of multiple human physiologic and disease states.
[00258] The intravenous route of administration corresponds to a possible human therapeutic route. The dose of the test articles was based on the results of the dose finding studies of analogous compounds previously performed in the African green monkey.
[00259] African green monkeys were chosen as the primate of choice as the test substances' target sequences are conserved across species with 100 % homology in primates.
Additionally, the test substance is a synthetic oligonucleotide. Consequently, dosing in primates allows for a superior assessment of the efficacy of these compounds that would be more reflective of the uptake likely to be seen in humans than in any other species.
ANIMALS
Species [00260] Chlorocebus sabaeus, non-human primate Breed [00261] African green monkey indigenous to St. Kitts.
Source [00262] RxGen, Lower Bourryeau, St. Kitts, West Indies.
Expected Age [00263] The test animals were adults.
Expected Body Weight [00264] The monkeys weigh approximately 3-4 kg. The actual range may vary but will be documented in the data.
Sex .. [00265] The test animals were adult females.
Number of Animals [00266] Ten animals were screened to ensure identification of 8 animals appropriate for enrollment in the study.
Number on Study [00267] Females: 8 Justification fbr Number on Study [00268] This study was designed to use the fewest number of animals possible, consistent with the primary objective of evaluating the therapeutic efficacy of the test article in the African green monkey and prior studies of the systemic administration of this type of oligonucleotide in this species.
Animal Specification 1002691 Ten adult African Green monkeys in the weight range of 3 to 4 kg, were employed in the study. The monkeys were drug-naïve adult animals humanely trapped from the feral population that inhabits the island. Trapped monkeys were treated with antihelminthics to eliminate any possible intestinal parasite burden and were observed in quarantine for a minimum of 4 weeks prior to screening for study enrollment. The age of trapped monkeys were estimated by size and dentation, with the exclusion of older animals from the study.
Prior to study enrollment, a clinical exam was performed on each monkey, including evaluation of locomotion and dexterity. Blood samples were taken and sent to Antech Diagnostics (Memphis, TN) for comprehensive clinical chemistries and a complete blood count and lipid profiles (see sections 9.2 and 319567928 for specifications).
Monkeys with abnormal lab values, as determined by comparison to the established normal range for monkeys in the St. Kitts colony, were excluded from the study. In order to identify 8 monkeys that satisfy this criterion, 10 monkeys were screened, with the screening of additional animals as needed. Before study initiation, the selected monkeys will be transferred to individual cages to acclimate to individual housing for a one-week period. Only animals deemed suitable for experimentation will be enrolled in the study. The actual (or estimated) age and weight ranges at the start of the study will be detailed in the raw data and final report.
Animal Health and Welfare [00270] The highest standards of animal welfare were followed and adhered to guidelines stipulated by the St. Kitts Department of Agriculture and the U.S. Department of Health and Human Services. All studies will be conducted in accordance with these requirements and all applicable codes of practice for the care and housing of laboratory animals.
All applicable standards for veterinary care, operation, and review as contained in the NIH
Guide for the Care and Use of Animals. The St. Kitts facility maintains an animal research committee that reviews the protocols and inspects the facilities as required by the Guide.
The Foundation has an approved assurance filed with the Office of Laboratory Animal Welfare, as required by the Guide, #A4384-01 (Axion Research Foundation/St. Kitts Biomedical Foundation).
There are no special nonhuman primate veterinary care issues and biohazard issues raised by the research specified in this study.
Housing and Environment [00271] To allow detection of any treatment-related clinical signs, the animals were housed individually prior to surgery and postoperatively until sacrifice. The primate building in which the individual cages were situated were illuminated entirely by ambient light, which at 17 degrees north latitude approximates a 12hr:12hr light-dark cycle as recommended in the U.S.
D.H.H.S guidelines. The RxGen primate building was completely ventilated to the outside.
Additional air movement was assured by ceiling fans to maintain a constant target temperature of 23-35 C, as is typical of St. Kitts throughout the year. Twenty-four hour extremes of temperature and relative humidity (which also will not be controlled) were measured daily. During the study, the cages were cleaned at regular intervals.
Diet and Water [00272] Each animal was offered approximately 90 grams per day of a standard monkey chow diet (TekLad, Madison, WI). The specific nutritional composition of the diet was recorded. The water was periodically analyzed for microbiological purity. The criteria for acceptable levels of contaminants in stock diet and water supply were within the analytical specifications established by the diet manufacturer and the periodic facility water evaluations, respectively. The water met all criteria necessary for certification as acceptable for human consumption.
EXPERIMENTAL DESIGN
Animal Identification and Randomization [00273] Allocation was done by means of a stratified randomization procedure based on bodyweight and plasma cholesterol profiles. Prior to and after allocation to a group, each animal was identified by a tattoo on the abdomen. Tattoos are placed on all colony animals as a means of identification in the course of routine health inspections. A cage plan was drawn up to identify the individuals housed within, and individual monkeys were further identified by a labeled tag attached to their respective cage. Group sizes, doses and identification numbers [00274] The animals were assigned to 2 treatment groups, comprised of 4 monkeys in each group. Specific animal identification numbers were provided to each monkey according to the facility numbering system. This system uniquely identifies each monkey by a letter followed by a three digit number, e.g. Y032.
Route and Frequency of Administration [00275] Animals were dosed once daily on Days 1, 3, and 5 delivered intravenously by manual infusion over ¨10 min. The infusion rate will be 24 mL/kg/h. The animals were sedated with ketamine and xylazine prior to and during the dosing procedure. A
venous catheter (Terumo mini vein infusion set, 20 gauge needle, or similar appropriate infusion set) was inserted into the saphenous vein. Dosing took place in each monkey between 8:00 and 10:00 a.m. shortly after the animals wake and prior to feeding. A blood sample to assess plasma cholesterol and other lipid levels as described in Blood Chemistry section below, was collected just prior to each infusion. Blood collection preceded feeding at both sampling intervals to minimize dietary effects on cholesterol measurements.
Clinical Observations [00276] All visible signs of reaction to treatment were recorded on each day of dosing. In addition, the animals were examined at least once each week for physical attributes such as appearance and general condition.
Body Weights [00277] Body weights were recorded at weekly intervals during the treatment and post-treatment periods.
Food Consumption 1002781 Individual food consumption was not quantified. Feeding patterns were however monitored and a note made of any major changes.
Mortality and Morbidity 1002791 Mortality and morbidity will be recorded. Any decision regarding premature sacrifice will be made after consultation with the Study Director and with the Sponsor's Monitoring Scientist. if possible. Animals that are found dead or killed prematurely will be subjected to necropsy with collection of liver, kidney, heart and spleen lung tissues for histopathology. In the event of premature sacrifice a blood sample will also be taken (if possible) and the parameters determined. Animals that are found dead after regular working hours will be refrigerated overnight and necropsies performed at the start of' the next working day. If the condition of an animal requires premature sacrifice, it will be euthanized by intravenous overdose of sodium pentobarbital. All research is governed by the Principles for Use of Animals. RxGen is required by law to comply with the U.S. Department of Health and Human Services standards for primate facility, which dictates the levels of severity that the procedures within this study. specified as mild, must abide.
CLINICAL LABORATORY STUDIES
Fat Biopsies 1002801 A subcutaneous fat biopsy was performed on all study monkeys except Y775 on study days 26 by tissue extraction through a 1 cm midlinc incision inferior to the umbilicus.
TM
Biopsies were immediately immersed in a labeled cryonabe containing 2 inls of RNAlater TM
(Qiagen) and incubated at 4 C overnight, after which the RNAlater was aspirated and the sample tube flash frozen in liquid nitrogen. Following transportation in liquid nitrogen total RNA as isolated for real-time qPCR of target genes.
Rau/is:
1002811 Real time PCR results show an increase in SIRTI mRNA levels in fat biopsies from monkeys dosed with CUR-963, an oligonueleotide designed to SIRTI
antiscnse CV396200.I. compared to monkeys dosed with CUR-962 (SEQ ID NO.:53 ). alt oligonucleotide which had no effect on SIRTI expression in vitro (designed to ApoA I
antisense DA327409, data not shown). mRNA levels were determined by real time PCR
(Figure 4).
1002821 Although the invention has been illustrated and described with respect to one or more implementations. equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.
1002831 The Abstract of the disclosure will allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the following claims.
[00165] Specific examples of some preferred oligonucleotides envisioned for this invention include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2 --NH--0--CH2, CH,--N(CH3)--0--CH2 [known as a methylene(methylimino) or MMI backbone], CH2 --0--N (CH3)--CH2, CH2 ¨N (CH3)--N
(CH3)--CH2 and 0--N (CH3)--CH2 --CH2 backbones, wherein the native phosphodiester backbone is represented as 0--P--0--CH,). The amide backbones disclosed by De Mesmaeker et at. (1995) Acc. Chem. Res. 28:366-374 are also preferred. Also preferred are oligonucleotides having morpholino backbone structures (Summerton and Weller, U.S. Pat.
No. 5,034,506). In other preferred embodiments, such as the peptide nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al. (1991) Science 254, 1497). Oligonucleotides may also comprise one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 0(CH2)n CH3, 0(CH2)n NH2 or 0(CH2)n CH3 where n is from 1 to about 10; Cl to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3 ;
OCF3; 0--, S--, or N-alkyl; S--, or N-alkenyl; SOCH3; SO2 CH3; 0NO2; NO2; N3; NH2;
heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;
substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy [2'-0-CH2 CH2 OCH3, also known as 2'-0-(2-methoxyethyl)] (Martin et at., (1995) Hely. Chim. Acta, 78, 486). Other preferred modifications include 2'-methoxy (2'-0--CH3), 2'- propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
[00166] Oligonucleotides may also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleotides include nucleotides found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5- hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleotides, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5- bromouracil, hydroxymethyluracil, 8-azaguaninc, 7-dcazaguaninc, N6 (6-aminohcxyl)adcninc and 2,6-diaminopurine. (Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp75-77; Gebeyehu, G., (1987) etal. Nucl. Acids Res. 15:4513). A "universal"
base known in the art, e.g., inosine, may be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 C. (Sanghvi, Y. S., in Crooke, S. T.
and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.
276-278) and are presently preferred base substitutions.
[00167] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety (Letsinger et at., (1989) Proc.
Natl. Acad. Sci. USA 86, 6553), cholic acid (Manoharan et at. (1994) Bioorg.
Med. Chem. Let.
4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et at. (1992) Ann.
NY. Acad. Sci.
660, 306; Manoharan et at. (1993) Bioorg. Med. Chem. Let. 3, 2765), a thiocholesterol (Oberhauser et at., (1992) Nucl. Acids Res. 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et at. EMBO J. 1991, 10, 111; Kabanov et at. (1990) FEBS Lett. 259, 327; Svinarchuk et at. (1993) Biochimie 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero- 3-H-phosphonate (Manoharan etal. (1995) Tetrahedron Lett. 36, 3651; Shea etal.
(1990) Nucl.
Acids Res. 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et at. (1995) Nucleosides & Nucleotides, 14, 969), or adamantane acetic acid (Manoharan et al. (1995) Tetrahedron Lett. 36, 3651). Oligonucicotidcs comprising lipophilic moieties, and methods for preparing such oligonucleotides are known in the art, for example, U.S.
Pat. Nos.
5,138,045, 5,218,105 and 5,459,255.
[00168] It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleotide within an oligonucleotide.
The present invention also includes oligonucleotides which are chimeric oligonucleotides as hereinbefore defined.
[00169] In another embodiment, the nucleic acid molecule of the present invention is conjugated with another moiety including but not limited to abasic nucleotides, polyether, polyamine, polyamides, peptides, carbohydrates, lipid, or polyhydrocarbon compounds.
Those skilled in the art will recognize that these molecules can be linked to one or more of .. any nucleotides comprising the nucleic acid molecule at several positions on the sugar, base or phosphate group.
1001701 The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of one of ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling VA) to synthesize fluorescently labeled, biotinylated or other modified oligonucleotides such as cholesterol-modified oligonucleotides.
[00171] In accordance with the invention, use of modifications such as the use of LNA
monomers to enhance the potency, specificity and duration of action and broaden the routes of administration of oligonucleotides comprised of current chemistries such as MOE, ANA, FANA, PS etc (ref: Recent advances in the medical chemistry of antisense oligonucleotide by Uhlman, (2000) Current Opinions in Drug Discovety & Development Vol 3 No 2).
This can be achieved by substituting some of the monomers in the current oligonucleotides by LNA
monomers. The LNA modified oligonucleotide may have a size similar to the parent compound or may be larger or preferably smaller. It is preferred that such LNA-modified oligonucleotides contain less than about 70%, more preferably less than about 60%, most preferably less than about 50% LNA monomers and that their sizes arc between about 5 and 25 nucleotides, more preferably between about 12 and 20 nucleotides.
1001721 Preferred modified oligonueleotide backbones comprise, but not limited to, phosphorothioates. chiral phosphorothioates. phosphorodithioatcs.
phosphotriesters, aminoalkylphosphotriesters. methyl and other alkyl phosphonates comprising l'alkylene phosphonates and chiral phosphonatcs, phosphinates. phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramiclates, thionophosphoramidates.
thionoalkylphosphonates. thionoalkylphosphotriesters, and boranophosphates having nomial 3'-5' linkages. 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleotide units are linked 3*-5' to 5'-3' or 2'-S' to 5'-2'. Various salts, mixed salts and free acid forms arc also included.
1001731 .. Representative United States patents that teach the preparation of the above phosphorus containing linkages comprise, but arc not limited to, US patent nos. 3,687,808;
4,469,863; 4,476,301; 5,023.243; 5. 177,196: 5,188,897; 5.264.423: 5,276.019;
5,278,302;
5,286,717; 5,321,131; 5,399,676: 5,405,939: 5,453,496: 5,455, 233: 5,466,677;
5,476.925;
5.519,126; 5,536,821; 5.541,306; 5.550.111; 5,563, 253; 5,571,799; 5.587,361;
and 5,625.050.
1001741 Preferred modified oligonueleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part From the sugar portion of a nucleotide); siloxatte backbones; sulfide, sulfoxide and sulfone backbones;
folmacetyl and thiofonnacetyl backbones; methylene formacetyl and thioformacetyl backbones;
alkene containing backbones; stilt-mime backbones. methyleneimino and inethylenehydrazino backbones: sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and C1-12 component parts.
1001751 Representative United States patents that teach the preparation of' the above oligonucleosides comprise, but are not limited to. US patent nos. 5,034,506;
5,166.315:
5.185,444; 5,214,134; 5,216,141: 5,235.033: 5.264, 562; 5, 264,564; 5,405.938;
5,434.257;
5,466,677; 5,470,967; 5,489,677; 5,541.307; 5,561,225; 5,596, 086; 5.602.240;
5,610,289;
5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5.663.312: 5.633,360;
5,677,437;
and 5,677,439.
1001761 In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an olig,onticleolide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are retained and are bound .. directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
Representative United States patents that teach the preparation of PNA
compounds comprise, but are not limited to, US patent nos. 5,539,082: 5,714,331: and 5,719,262.
Further teaching of PNA compounds can be found in o Nielsen et at.. Science, 1991, 254. 1497-1500.
1091771 In another preferred embodiment of the invention the oligonucleoticles with phosphorothioate backbones and oligonucicosides with heteroatom backbones, and in particular- C1-12-NH-O-CH2-,-CH2-N (C1-13)-0-C1-12-known us a methylene (methylimino) or MM1 backbone.- CH2-0-N (CH3)-CH2-.-CH2N(CH3)-N(CH3) CH2-and-O-N(CH3)-CH2-CH2- wherein the native phosphodiester backbone is represented as-O-P-0-0-12-of the above referenced US patent no. 5,489.677. and the amide backbones of the above referenced US patent no. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced US patent no. 5.034,506.
101781 Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; I':
0-. S-, or N-alkyl: 0-. S-. or N-alkenyl: 0-, S-or N-alkynyl: or 0 alkyl-0-alkyl. wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C to CO alkyl or C2 to CO
alkenyl and alkynyl. Particularly preferred are 0 (CI12)n OinC113. 0(012)n,OCI-13, 0(a-12)11Na-12. 0(012)nC113, 0(('112.)nONH2. and 0(CH2nON(CH2)ncH3)2 where n and m can be from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C to CO. (lower alkyl, substituted lower alkyl, alkaryl.
aralkyl, 0-alkaryl or 0-aralkyl, SH, SCH3. OCN. Cl, Br. CN, CF3, OCF3, SOCI13. SO2C1-13. 0NO2. NO2, N3, NH2, heterocycloalkyl, hetcrocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl. an RNA cleaving group, a reporter group. an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification comprises 2'-methoxyethoxy (2'-0-CH2CF1200-13. also known as 2'4)-(2- methoxyethyl) or 2'-M0E) (Martin et al., (1995) Halt'. Chun.
Acta, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification comprises 2'-dimethylaminooxyethoxy, i.e. , a 0(CH2)20N(C1-13)2 group, also known as 2'-DMA0E, as -40.
described in examples herein below, and 2'- climethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or DMAEOE:), i.e.. 2'-0-0-12-0-CH2-N
(CH2)2.
1001791 Other preferred modifications comprise 2'-methoxy (2'-0 CH3), 2'-aminopropoxy (2'-O CI 12C1-12042N112) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar miluctics such as cyclobutyl moieties in place of the pentofuntnosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures comprise, but are not limited to, US patent nos.
4,981.957: 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5.446,137; 5,466,786;
5.514, 785:
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873;
5,616, 265; 5,658,873; 5.670.633: and 5,700,920.
1001801 Oligonucleotides may also comprise nucleobase (often referred to in the art simply IS as "base") modifications or substitutions. As used herein, "unmodified"
or "natural"
nucleotides comprise the purine bases adenine (A) and guanine (G), and the pyrimidinc bases thymine (T), cytosine (C) and uracil (U). Modified nucleotides comprise other synthetic and natural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthinc, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine. 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo mei!, cytosine and thymine, 5-uracil (pseudo-uracil 4-thiouracil, 8-halo. 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-tritluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladeninc, 8-azaguanine and 8-azattdeninc, 7-dcazaguanine and 7-dcazatidenine and 3-dcazaguaninc and 3-dcazaadenine.
1001811 Further, nucleotides commise those disclosed in United States Patent No.
3.687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science And Engineering', pages 858-859, Kroschwitz, J.1., ed. John Wiley & Sons, 1990, those disclosed by Englisch et at,'Angewandle Chemie, International Edition', 1991, 30, page 613. and those disclosed by Sanghvi, Y.S.. Chapter 15. 'Antisense Research and Applications', pages 289-302, Crooke, S.T. and Lebleu. B. ca., CRC Press, 1993. Certain of these nucleotides are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These comprise 5-substituted pyrimidines, 6- azapyrirnidines and N-2. N-6 and 0-6 substituted purifies. comprising 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosinc. 5-methyleytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2cC (Sanahvi, V.S.. ('rooke, S.T. and Leblett, B., eds, 'Antisense Research and Applications', CRC Press, Boca Raton. 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-Omethoxyethyl sugar modifications.
1001821 Representative United States patents that teach the preparation of the above noted modified nucleotides as well as other modified nucleotides comprise, but are not limited to, US patent nos. 3,687.808, as well as 4,845.205; 5,130.302; 5,134,066; 5.175, 273; 5. 367,066;
5,431272: 5,457,187; 5.459.255; 5.484,908; 5,502,177; 5.525.711; 5,552.540;
5,587,469;
5.596.091; 5.614,617: 5,750,692, and 5,681,941.
1001831 Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotidc one or more moieties or conjugates, which enhance the activity.
cellular disnibution, or cellular uptake of the oligonucicotide.
1001841 Such moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et aL, Proc. Natl. .4cad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.. (1994) MmHg. Med. Client. Let., 4. 1053-1060). a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.. (1992) .4nn. N. )'. Acad. Sci.. 660, 306-309;
Manoharan et al..
(1993) Biooty. Med. ('hem. Let.. 3, 2765-2770). a thioeholesierol (Oberhauser ci at. (1992) ...his RCA., 20, 533-538), an aliphatic chain. e.g., dodecandiol or undecyl residues (Kabanov A. (1990) FESS Lett., 259, 327-330: Svinarchuk etal., (1993) Bioc.hiinie 75, 49-54), a phospholipid, e.g., di-hexadecyl-rue-glyeerol or triethylammonium 1.2-di-O-ltexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., (1995) Tetrahedron Len.. 36, 3651-3654;
Shea et al.. (1990) Nucl. Acids Res.. 18. 3777-3783). a polyamine or a polyethylene glycol chain (Maneharan er iiI.. (1995) Nucleosides & Nucleotides, 14, 969-973). or adamantane acetic acid (Manohartin et al.. (1995) Tetrahedron Lett., 36, 3651-3654), a palmityl moiety (Mishrti et al., (1995) Siochint. Biophys. Acta, 1264, 229-237), or an octadecylamine or hexylarnino-carbonyl-t oxycholesterol moiety (Crooke et al., (1996) J.
Pharmacrit rho... 277, 923-937).
1001851 Representative United States patents that teach the preparation of such oligonucleotides conjugates comprise, but are not limited to. US patent nos.
4,828,979;
4,948.882; 5,218,105; 5,525,465; 5.541,313; 5,545.730: 5,552. 538; 5.578,717.
5,580,731;
5,580.731; 5,591,584; 5,109,124; 5,118,802, 5,138,045; 5.414.077: 5.486, 603;
5,512,439;
5.578,718; 5,608,046; 4.587,044; 4,605,735; 4.667,025; 4,762, 779; 4.789,737;
4,824,941:
4.835.263; 4,876,335; 4.904,582; 4,958,013; 5,082, 830; 5,112.963: 5.214,136;
5,082,830;
5,112.963; 5.214,136; 5. 245.022: 5,254.469: 5.258,506; 5,262,536: 5.272,250;
5,292,873;
5,317.098; 5.371,241, 5,391, 723: 5,416.203, 5,451,463; 5,510,475: 5.512.667;
5,514.785; 5, 565,552; 5,567,810; 5,574,142; 5,585,481; 5.587.371; 5.595,726: 5,597,696;
5.599,923;
5,599, 928 and 5,688,941.
1001861 DIV; diseoveiy: The compounds of the present invention can also be applied in the areas of drug discovery and target validation. Tie present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between Sirtuin 1 (SIRT1) polynucleotides and a disease state, phenotype, or condition. These methods include detecting or modulating Sirtuin (SIRT1) polynucleotides comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of Sirtuin (SIRT I ) polynucleotides and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.
Assessing Up-regulation or Inhibition of Gene Expression:
1001871 Transfer of an exogenous nucleic acid into a host cell or organism can be assessed by directly detecting the presence of the nucleic acid in the cell or organism. Such detection can be achieved by several methods well known in the art. For example, the presence of the exogenous nucleic acid can be detected by Southern blot or by a polymerase chain reaction (PCR) technique using primers that specifically amplify nucleotide sequences associated with the nucleic acid. Expression of the exogenous nucleic acids can also be measured using conventional methods including gene expression analysis. For instance, mRNA
produced from an exogenous nucleic acid can be detected and quantified using a Northern blot and reverse transcription PCR (RT-PCR).
1001881 Expression of RNA from the exogenous nucleic acid can also be detected by measuring an enzymatic activity or a reporter protein activity. For example, antisense modulatory activity can be measured indirectly as a decrease or increase in target nucleic acid expression as an indication that the exogenous nucleic acid is producing the effector RNA.
Based on sequence conservation, primers can be designed and used to amplify coding regions of the target genes. Initially, the most highly expressed coding region from each gene can be used to build a model control gene, although any coding or non coding region can be used.
Each control gene is assembled by inserting each coding region between a reporter coding region and its poly(A) signal. These plasmids would produce an mRNA with a reporter gene in the upstream portion of the gene and a potential RNAi target in the 3' non-coding region.
The effectiveness of individual antisense oligonucleotides would be assayed by modulation of the reporter gene. Reporter genes useful in the methods of the present invention include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), antibiotic resistance determination.
Kits, Research Reagents, Diagnostics, and Therapeutics [00189] The compounds of the present invention can be utilized for diagnostics, therapeutics, and prophylaxis, and as research reagents and components of kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
[00190] For use in kits and diagnostics and in various biological systems, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, are useful as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.
[00191] As used herein the term "biological system" or "system" is defined as any organism, cell, cell culture or tissue that expresses, or is made competent to express products of the Sirtuin 1 (SIRT1) genes. These include, but are not limited to, humans, transgenic animals, cells, cell cultures, tissues, xenografts, transplants and combinations thereof.
[00192] As one non limiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, .. cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds that affect expression patterns.
[00193] Examples of methods of gene expression analysis known in the art include DNA
arrays or microarrays (Brazma and Vilo, (2000) FEBS Lett., 480, 17-24; Celis, et al., (2000) FEBS Lett., 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., (2000) Drug Discov. Today, 5, 415- 425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, (1999) Methods Enzytnol., 303, 258-72), TOGA
(total gene expression analysis) (Sutcliffe, et al., (2000) Proc. Natl. Acad. Sci. U.S.A., 97, 1976-81), protein arrays and proteomics (Celis, et al., (2000) FEBS Lett., 480, 2-16;
Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., (2000) Anal. Biochem. 286, 91-98;
Larson, et al., (2000) Cytometty 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, (2000) Cum Opin. Microbiol. 3, 316-21), comparative genomic hybridization (Carulli, et al., (1998) J. Cell Biochent. Suppl., 31, 286-96), FISH
(fluorescent in situ hybridization) techniques (Going and Gusterson, (1999) Eur. J. Cancer, 35, 1895-904) and mass spectrometry methods (To, Comb. (2000) Chem. High Throughput Screen, 3, 235-41).
[00194] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Sirtuin 1 (SIRT1). For example, oligonucleotides that hybridize with such efficiency and under such conditions as disclosed herein as to be effective Sirtuin 1 (SIRT1) modulators are effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding Sirtuin 1 (SIRT1) and in the amplification of said nucleic acid molecules for detection or for use in further studies of Sirtuin 1 (SIRT1). Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding Sirtuin 1 (SIRT1) can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabeling of the oligonueleotide, or any other suitable detection means. Kits using such detection means for detecting the level of Sirtuin 1 (SIRT1) in a sample may also be prepared.
[00195] The specificity and sensitivity of antisense are also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
[00196] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Sirtuin 1 (SIRT1) polynucleotides is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of Sirtuin 1 (SIRT1) modulator. The Sirtuin 1 (SIRT1) modulators of the present invention effectively modulate the activity of the Sirtuin 1 (SIRT1) protein or modulate the expression of the Sirtuin 1 (SIRT1) protein. In one embodiment, the activity or expression of Sirtuin 1 (SIRT1) in an animal is inhibited by about 10% as compared to a control. Preferably, the activity or expression of Sirtuin 1 (SIRT1) in an animal is inhibited by about 30%. More preferably, the activity or expression of Sirtuin 1 (SIRT1) in an animal is inhibited by 50%
or more. Thus, the oligomeric compounds modulate expression of Sirtuin 1 (SIRT1) mRNA by at least 10%, by at least 50%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100% as compared to a control.
[00197] In one embodiment, the activity or expression of Sirtuin 1 (SIRT1) and/or in an animal is increased by about 10% as compared to a control. Preferably, the activity or expression of Sirtuin 1 (SIRT1) in an animal is increased by about 30%. More preferably, the activity or expression of Sirtuin 1 (SIRT1) in an animal is increased by 50%
or more. Thus, the oligomeric compounds modulate expression of Sirtuin 1 (SIRT1) mRNA by at least 10%, by at least 50%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100% as compared to a control.
[00198] For example, the reduction of the expression of Sirtuin 1 (SIRT1) may be measured in serum, blood, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids. tissues or organs being analyzed contain a nucleic acid molecule encoding Sirtuin 1 (SIRT1) peptides and/or the Sirtuin 1 (S I RT I ) protein itself.
1001991 The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.
Conjugates 1002001 Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleolide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols. polyethers, groups that enhance the phamtacodynamic properties of limners, and groups that enhance the pharmacokinetic properties of oligomers. Typicalconjugatc groups include cholesterols, lipids, phospholipids.
biotin, phenazine, folate, phenanthridine. anthraquinone, acridine, fluorescents, rhodamines, coumarins. and dyes. Groups that enhance the pharmacodynamie properties, in the context of this invention, include groups that improve uptake. enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
Groups that enhance the pharmacokinetic properties. in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention.
Representative conjugate groups arc disclosed in International Patent Application No.
PC'T!US92109196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,1(60.
Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioethcr, e.g., hexy1-5- tritylthiol. a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glyeero-3-Flphosphonate. a polyamine or a polyethylene glycol chain, or adamantanc acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example.
aspirin, warfarin, phenylbutazone. ibuprofen. suprofen. fenbufen, ketoprolen, (S)-(+)-pranoprofen, carpro fen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
1002011 Representative United States patents that teach the preparation of such oligonucicotides conjugates include, but arc not limited to. U.S. Pat. Nos.
4.828,979:
$ 4.948,882: 5,218,105; 5,525,465; 5,541,313; 5,545.730; 5,552,538;
5,578.717, 5,580,731:
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;
5,512,439;
5.578,718; 5,608,046; 4,587,044; 4,605.735; 4,667.025; 4,762.779; 4,789,737:
4,824,941:
4.835,263; 4,876,335; 4,904,582; 4,958,013; 5,082.830; 5,112,963; 5,214,136;
5.082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250:
5.292.873;
5,317.098: 5,371,241, 5,391,723; 5,416,203, 5.451,463; 5,510,475; 5,512.667;
5,514,785;
5,565.552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5.595,726; 5,597,696:
5.599.923;
5,599.928 and 5,688.941.
FormulaiionA
1002021 The compounds of the invention may also be admixed. encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as lot-example, liposomes, receptor-targeted molecules. oral, rectal, topical or other formulations, for assisting in uptake. distribution andior absorption.
Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos.
5,108.921; 5.354,844:
5.416,016: 5,459,127; 5.521,291; 5,543,165: 5.547,932; 5,583.020; 5,591,721:
4.426,330;
4,534,899: 5,013,556; 5.108,921; 5,213,804; 5,227,170; 5.264.221; 5,356,633;
5,395,619;
5,416,016; 5,417,978; 5,462,854: 5,469,854; 5,512,295: 5.527,528; 5.534,259:
5,543,152:
5,556.948; 5,580,575; and 5,595,756.
1002031 Although, the antisense oligonucleotides do not need to be administered in the context of a vector in order to modulate a target expression and/or function, embodiments of the invention relates to expression vector constructs for the expression of antisense olig,onueleotides, comprising promoters, hybrid promoter gene sequences and possess a strong constitutive promoter activity, or a promoter activity which can be induced in the desired case.
1002941 In an embodiment. invention practice involves administering at least one of the foregoing antisense oligonucleotides with a suitable nucleic acid delivery system. In one embodiment, that system includes a non-viral vector operably linked to the polynucleotide.
Examples of' such nonviral vectors include the oligonucleotidc alone (e.g. any one or more of SEQ ID NOS: 7 to 48 or in combination with a suitable protein, polysaccharide or lipid formulation.
1002051 Additionally suitable nucleic acid delivery systems include viral vector, typically sequence from at least one of an adenovirus, adenovirus-associated virus (AAV), helper-.5 dependent adenovirus, retrovirus, or hcmagglutinatin virus of Japan-liposome (HVJ) complex.
Preferably, the viral vector comprises a strong cukaryotic promoter operably linked to the polynucleotide e.g.. a cytomegalovirus (CMV) promoter.
1002061 Additionally preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney nutrine leukemia viruses and WV-based viruses One preferred HIV-based viral vector comprises at least two vectors wherein the gag and poi genes are from an HIV genomc and the env gene is from another virus.
DNA viral vectors arc preferred. These vectors include pox vectors such as orthopox or avipos vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector [Geller.
A.1. et al., (1995) J. Neurochent, 64: 487; Lim. F., etal., in DNA Cloning: Mammalian Systems, D.
Glover, Ed.
(Oxford Univ. Press. Oxford England) (1995); Geller, A.I. et al.,(1993) Proc Natl. Acad. Sc!.:
U.S.A.:90 7603: Geller, Al., etal.. (1990) Proc Natl. Acta Sc! USA: 87:11491, Adcnovirus Vectors [LeGal LaSalle et al.. Science, 259:988 (1993); Davidson. et al., (1993) A'at. Genet.
3: 219; Yang, etal.. (1995).!. Vim!. 69: 20041 and Adeno-associated Virus Vectors [Kapliit, M.G.. et a/.. (1994 ) Nat. Genet. 8:148].
1002071 The antisensc compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
1002081 The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.c., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonuelcotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Put. No. 6.287.860.
1002091 The present invention also includes pharmaceutical compositions and formulations that include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'-0-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful.
[00210] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
[00211] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
[00212] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.
[00213] Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 lam in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug that may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase.
Mieroemulsions are included as an embodiment of the present invention.
Emulsions and their uses are well known in the art and are further described in U.S. Pat. No.
6,287,860.
1002141 Formulations of the present invention include liposornal formulations. As used in the present invention, the term "liposorne" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes arc unilamellar or multilamellar vesicles which have a membrane formed From a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged arc believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
1002151 Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids. When incorporated into liposomcs, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomeslacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860.
1002161 The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products.
formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S.
Pat. No. 6,287.860.
1002171 In one embodiment. the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also .. enhance the pertneability of lipophilic drugs. Penetration enhancers may be cla.ssified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chclating nonsurfitctants. Penetration enhancers and their uses arc further described in U.S. Pat. No. 6,287,860.
1002181 One of skill in the art will recognize that formulations are routinely designed according to their intended use. i.e. route of administration.
1002191 Preferred formulations for topical administration include those in which the olionucleotides of the invention are in admixture with a topical delivery agent such as lipids.
liposomes, fatty acids, fatty acid esters, steroids, dictating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoyl-phosphatidyl DOPE
ethanolamine, dimyristoylphosphatidyl cholinc DMPC, distearolyphosphatidyl choline) negative (e.g.
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.
dioleoyltetrarnethylaminopropyl DOTAP and dioleoyl-phosphatidyl ethanolamine DOTMA).
1002201 For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters. pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860.
1002211 Compositions and formulations for oral administration include powders or granules, microparticulaies, nanoparticulates. suspensions or solutions in water or non-aqueous media. capsules, gel capsules. sachets, tablets or minitableis.
Thickeners, flavoring agents, diluents, emulsifiers. dispersing aids or binders may be desirable.
Preferred oral formulations are those in which oligonucleotides of' the invention are administered in conjunction with one or more penetration enhancers surfactants and dictators.
Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
Preferred bile acids/salts and fatty acids and their uses arc further described in U.S. Pat. No.
6,287,860. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids!salts. A
particularly preferred combination is the sodium salt of Laurie acid, capric acid and UDCA.
Further penetration enhancers include polyoxyethylene-9-lauryl ether.
polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles. or complexed to form micro or namoparticles.
Oligonucicotide complexing agents and their uses are further described in U.S.
Pat. No.
6,287.860.
1002221 Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to. penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
1002231 Certain embodiments of the invention provide pharmaceutical compositions containing one or more olicomeric compounds and one or more other chemotherapeutic agents that function by a non-antisense mechanism. Examples of' such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as datmorubiein.
datmomycin, dactinomycin, dosortibicin, epirubicin, idarubicin. esorubicin.
bleomycin.
mafosfitmide, ifosfitmide. cytosine arabinoside, bischloroethyl- nitrosurea, busulfitn.
mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention.
Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
[00224] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. For example, the first target may be a particular antisense sequence of Sirtuin 1 (SIRT1), and the second target may be a region from another nucleotide sequence. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same Sirtuin 1 (SIRT1) nucleic acid target. Numerous examples of antisense compounds are illustrated herein and others may be selected from among suitable compounds known in the art. Two or more combined compounds may be used together or sequentially.
Dosing:
[00225] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 pig to 100 g per kg of body weight, and may be given once or more daily. weekly. monthly or yearly, or even once every 2 to 20 years.
Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 lig to 100 g per kg of body weight. once or more daily. to once every years.
1002261 While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with 15 the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments.
By their citation of various references in this document. Applicants do not admit any particular reference is "prior art" to their invention.
Embodiments of inventive compositions and methods are illustrated in the following examples.
EXAMPLES
25 1002281 The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in pmportions and alternatives in elements of the components shown will be apparent to those skilled in the art and arc within the scope of embodiments of the present invention.
Evanple I: Design p/. antisense oligonucleothks specific ftir a nucleic acid puilecule 30 antisense anchor sense strand qrSirtuin I (S I RT1 ) polynticleolide 1002291 As indicated above the term "oligonucleotide specific for" or "oligonucleotide targets" refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (ii) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene.
[00230] Selection of appropriate oligonucleotides is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products.
Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity. These procedures allow the selection of oligoncueltodes that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species. One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.
[00231] An antisense compound is "specifically hybridizable" when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and/or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays [00232] The hybridization properties of the oligonucleotides described herein can be determined by one or more in vitro assays as known in the art. For example, the properties of the oligonucleotides described herein can be obtained by determination of binding strength between the target natural antisense and a potential drug molecules using melting curve assay.
[00233] The binding strength between the target natural antisense and a potential drug molecule (Molecule) can be estimated using any of the established methods of measuring the strength of intermolecular interactions, for example, a melting curve assay.
[00234] Melting curve assay determines the temperature at which a rapid transition from double-stranded to single-stranded conformation occurs for the natural antisense/Molecule complex. This temperature is widely accepted as a reliable measure of the interaction strength between the two molecules.
1002351 A melting curve assay can be performed using a eDNA copy of the actual natural antisense RNA molecule or a synthetic DNA or RNA nucleotide corresponding to the binding site of the Molecule. Multiple kits containing all necessary reagents to perform this assay arc TM
available (e.g. Applied Biosystems Inc. MeltDoctor kit). These kits include a suitable buffer solution containing one of the double strand DNA (dsDNA) binding dyes (such as ABI FIRM
dyes, SYBR Green, SYTO, etc.). The properties of the dsDNA dyes are such that they emit almost no fluorescence in free form, but are highly fluorescent when bound to dsDNA.
1002361 To perform the assay the cDNA or a corresponding oligonucleotide are mixed with Molecule in concentrations defined by the particular manufacturer's protocols.
The mixture is heated to 95 C to dissociate all pre-formed dsDNA complexes. then slowly cooled to room temperature or other lower temperature defined by the kit manufacturer to allow the DNA
molecules to anneal. The newly formed complexes arc then slowly heated to 95 C
with simultaneous continuous collection of data on the amount of fluorescence that is produced by the reaction. The fluorescence intensity is inversely proportional to the amounts of dsDNA
present in the reaction. The data can be collected using a real time PCR
instrument compatible TM TM
with the kit (e.g.AB1's StepOne Plus Real Time PCR System or LightTyper instrument.
Roche Diagnostics, Lewes, UK).
1002371 Melting peaks arc constructed by plotting the negative derivative of fluorescence with respect to temperature (-d(Fluorescence)idT) on the y-axis) against temperature (x-axis) TM
using appropriate software (for example LighiTyper (Roche) or SDS Dissociation Curve.
ABI). The data is analyzed to identify the temperature of the rapid transition from dsDNA
complex to single strand molecules. This temperature is called Tm and is directly proportional to the strength or intentction between the two molecules. Typically. Tin will exceed 40 'C'.
Example 2: Modulation ql mlynuckatide3 Materials and Methods:
Treatment M'IlepG2 cells will? naked amisense oligonueleotides:
1002381 I1ep02 cells from ATCC (cat# HB-8065) were grown in growth media (MENVEBSS (1-lyclone cat ;I-SI-130024. or Mediatech cat # MT-10-010-CV) +10%
1:13S
(Mediatech catO MT35- 011-CV) + penicillinistreptomycin (Mediatech cat# MT30-002-CI)) at 37cC and 5% CO2. One day before the experiment the cells were replated at the density of 0.5 x 1 Osiml into 6 well plates and incubated at 37 C and 5% CO2. On the day of the experiment the media in the 6 well plates was replaced with 1.5 mitwell of fresh growth media. All antisense oligonucleotides were diluted in water to the concentration of 20 pM. 2 pl of this solution was incubated with 400 pl of Opti-MEM media (Gibco cat#31985-070) and TM
4 ul of Lipofcctamine 2000 (Invitroaen cart/ 11668019) at room temperature for 20 min and applied to each well of the 6 well plates with HcpG2 cells. Similar mixture including 2 IA of water instead of the oligonucleotide solution was used for the mock-transfected controls.
After 3-18 h of incubation at 37 C and 5% CO2 the media was changed to fresh growth media. 72 It after addition of antisense oligonucleotides the cells were redosed as described in above. 48h after the second addition of antisense oligonucleotides the media was removed and RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat #
TM
Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cats 74181) Following the manufacturers' instructions. 600 lig of RNA was added to the reverse transcription reaction performed using Verso cDNA kit from Thermo Scientific (ctit#AB1453B) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to TM
monitor gene expression by real time PCR using ABI 'ragman Gene Expression Mix TM
(cat#4369510) and primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00202021...m1 by Applied Biosystems Inc.. Foster City CA).
The following PCR cycle was used: 50 C' for 2 min. 95 C for 10 min, 40 cycles of (95 C for 15 seconds, 60 C for I min) using StcpOne Plus Real Time PCR Machine (Applied Biosystems).
Fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in 'SS-normalized dCt values between treated and mock-transfectcd samples.
Results:
1002391 Real time PCR results show that the levels of the SIRTI mRNA in HepG2 cells significantly increased 48 h after treatment with some antisense oligonucleotides to SIRTI
antisense CV396200 (Figure 2. 3A).
1002401 Real Time PCR results show that levels of SIRTI mRNA in 1lepG2 cells are significantly increased in one of the oligonucleotides designed to SIRTI
antisense CV396200.
The burs denoted us CUR- 1230. CUR- 123 1, CUR- 1 232 and CUR-1230 correspond to SEQ
ID NOs:33-36. (Fig 6) 1002411 Real Time PCR results show that levels of SIRTI mRNA in HepG2 cells are significantly increased in two of the oligonucleotides designed to SIRTI
antisense CV428275.
The bars denoted as CUR-1302, CUR-1304, CUR- 1303 and CUR-I305 correspond to SEQ
ID NOs:37-40. (Fig 7) 1002421 The results show that a significant increase in SIRTI mRNA levels in HepG2 cells 48 hours after treatment with one of the oligonucleotides designed to SIRT
antisense BE7I7453. The burs denoted as CUR-I264, CUR1265 and CUR-1266 correspond to SEQ
ID
NOs.: 41-43 respectively. (Fig 8) 1002431 The results show that the levels of the SIRT1 mRNA in FlepG2 cells are significantly increased 48 h after treatment with three of the oligonucicotides designed to sum antisense AV7I 8812. The bars denoted as CUR-I294, CUR-1297, CUR-1295, CUR-1296 and CUR-I298 correspond to SEQ ID NOs.: 44-48 respectively. (Fig 9) Treatment qf Vero76 cells with antisense oligonuclemides:
1002441 Vcro76 cells from ATCC (cat# CRL-1587) were grown in growth media (MEM'EBSS (Hyclone cat #SH30024. or Nlediatech cat # MT-10-010-CV) -10% FBS
(Mediated) cat# 011-CV)+ penicillin/streptomycin (Mediatech cut;! W30-0024.1)) at 37 C and 5% CO2. One day before the experiment the cells were replated at the density of 1.5 105/m1 into 6 well plates and incubated at 37 C and 5% CO2. On the day of the experiment the media in the 6 well plates was changed to fresh growth media. All antisense oligonucicotides were diluted in water to the concentration of 20 1.1114. 2 pl of this solution was incubated with 400 III of Opti-MEM media (Gibco cat431985-070) and 4 ul of Lipofcetamine 2000 (Invitrogen cat# 11668019) at room temperature for 20 min and applied to each well of the 6 well plates with Ven)76 cells. Similar mixture including 2 Id of water instead of the oligonucleotide solution was used for the mock- transfected controls. After 3-18 h of incubation at 37 C and 5% CO2 the media was changed to fresh growth media. 48 h after addition of antisense oligonucleotidcs the media was removed and RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat # 2.3105) or RNeasy Total RNA Isolation kit from Qiagcn (cat# 74181), following the manufacturers instructions. 600 ng of RNA was added to the reverse transcription reaction performed using Verso cDNA kit from Themo Scientific (cat#AB1453B) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PCR using A131 Taqmun Gene EXIMS:ii011 Mix (cab14369510) and primers/probes designed by ABI (Applied Biosystems Tagman Gene Expression Assay:
Hs00202021_ml by Applied Biosystems Inc.. Foster City CA). The following PCR cycle was used: 50 C for 2 9.5 C for 10 mitt. 40 cycles of (95 C for IS seconds. 60 C for I min) using StepOne Plus Real Time PCR Machine (Applied Biosystems). Fold change in gene expression after treatment with antisensc oligonucleolides was calculated based on the difference in I8S-normalized dCt values between treated and mock-transfected samples.
Results:
[00245] Real time PCR results show that the levels of the SIRT1 mRNA in Vero cells significantly increased 48 h after treatment with antisense oligonucleotides to SIRT1 antisense CV396200 (Figure 3B).
Example 3: Modulation of SIR Ti Gene Expression Materials and Methods Treatment of HepG2 cells with naked antisense oligonucleotides:
[00246] HepG2 cells from ATCC (cat# HB-8065) were grown in growth media (MEM/EBSS (Hyclone cat #SH30024, or Mediatech cat # MT-10-010-CV) +10% FBS
(Mediatech cat# MT35- 011-CV) + penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37 C and 5% CO2. One day before the experiment the cells were replated at the density of 0.5 x 105/m1 into 6 well plates and incubated at 37 C and 5% CO2. On the day of the experiment the media in the 6 well plates was replaced with 1.5 ml/well of fresh growth media. All antisense oligonucleotides were diluted in water to the concentration of 20 [tM. 2 111 of this solution was incubated with 400 pi of Opti-MEM media (Gibco cat#31985-070) and 4 ul of Lipofectamine 2000 (Invitrogen cat# 11668019) at room temperature for 20 min and applied to each well of the 6 well plates with HepG2 cells. Similar mixture including 2 iõt1 of water instead of the oligonucleotide solution was used for the mock-transfected controls.
After 3-18 h of incubation at 37 C and 5% CO2 the media was changed to fresh growth media. 72 h after addition of antisense oligonucleotides the cells were redosed as described in above. 48h after the second dosing of antisense oligonucleotides the media was removed and RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat #
Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat# 74181) following the manufacturers' instructions. 600 ng of RNA was added to the reverse transcription reaction .. performed using Verso cDNA kit from Thermo Scientific (cat#AB1453B) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PCR using AB1 Taqman Gene Expression Mix (cat#4369510) and primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00202021_m 1 by Applied Biosystems Inc., Foster City CA).
The following PCR cycle was used: 50 C for 2 min, 95 C for 10 min, 40 cycles of (95 C for 15 seconds, 60 C for 1 min) using StepOne Plus Real Time PCR Machine (Applied Biosystems).
Fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in 18S-normalized dCt values between treated and mock-transfected samples.
[002471 Primers and probe for the custom designed Taqman assay for exon 4:
AACTGGAGCTGGGGTGTCTGTTTCA (SEQ ID NO:49) of the SIRT1 natural antisense CV396200.
Forward Primer Seq. CCATCAGACGACATCCCTTAACAAA (SEQ ID NO: 50) Reverse Primer Seq. ACATTATATCATAGCTCCTAAAGGAGATGCA (SEQ ID NO: 51) Reporter Seq. CAGAGTTTCAATTCCC (SEQ ID NO: 52) Results:
1002481 The results show that the levels of the SIRT1 mRNA in HepG2 cells are significantly increased 48 h after treatment with one of the siRNAs designed to sirtas (sirtas_5, P=0.01). In the same samples the levels of sirtas RNA were significantly decreased after treatment with sirtas 5, but unchanged after treatment with sirtas_6 and sirtas 7, which also had no effect on the SIRT1 mRNA levels (Fig. 1B). sirtas 5, sirtas_6 and sirtas_7 correspond to SEQ ID NO: 30, 31 and 32 respectively.
Treatment of primary monkey hepatoeytes.
1002491 Primary monkey hepatocytes were introduced into culture by RxGen Inc.
and plated in 6 well plates. They were treated with oligonucleotides as follows.
The media in the 6 well plates was changed to fresh growth media consisting of William's Medium E
(Sigma cat#W4128) supplemented with 5% 113S, 50 U/ml penicillin and 50 ug/m1 streptomycin, 4 ug/ml insulin, 1 uM dexamethasone, 10 ug/ml Fungin (InVivogen, San Diego CA).
All antisense oligonucleotides were diluted to the concentration of 20 p.M. 2 [d of this solution was incubated with 400 il of Opti-MEM media (Gibco cat#31985-070) and 4 pl of Lipofectamine 2000 (Invitrogen cat# 11668019) at room temperature for 20 min and applied to each well of the 6 well plates with cells. Similar mixture including 2 111 of water instead of the oligonucleotide solution was used for the mock-transfected controls. After 3-18 h of incubation at 37 C and 5% CO2 the media was changed to fresh growth media. 48 h after addition of antisense oligonucleotides the media was removed and RNA was extracted from the cells using S V Total RNA Isolation System from Promega (cat # Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat# 74181) following the manufacturers' instructions. 600 ng of RNA was added to the reverse transcription reaction performed using Verso cDNA kit from Thermo Scientific (cat#AB1453B) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PCR using ABI Taqman Gene Expression Mix (cat#4369510) and primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay:
Hs00978340_ml by Applied Biosystems Inc., Foster City CA). The following PCR cycle was used: 50 C for 2 min, 95 C for 10 min, 40 cycles of (95 C for 15 seconds, 60 C for I min) using Mx4000 thermal cycler (Stratagem). Fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in NS-normalized en values between treated and mock-transfectcd samples.
Results:
1002501 The results are shown in Figure. 5. Real time PCR results show an increase in SIRTI mRNA levels after treatment with an oligonueleotide against S1RT I
antisense.
to Example 4: Efficacy and Duration 44ction Study ocCUR 96$ in the African Green llonkey 1002511 The objective of this study was to assess and compare the effect of antisense knockdown of the discordant noncoding antisense sequences that regulate the SIRT I genes following intravenous administration in a nonhuman primate model. The antisense oligonucicotide test articles designed to inhibit the SIRTI regulatory sequences were designated as CUR 963.
CUR 963: +G* T*C*T*G*A*T*G*G*-FA*.t.G*+A (SEQ ID N(); 26).
CUR 962 (control): 4-G*+C*T*A*G*T*C*T*G++T*-rf*-1-G (SEQ ID NO:53).
REGULATORY TEST GUIDELINES
1002521 This study was designed in accordance with accepted toxicological principles and to comply with International Conference of Harmonization (ICH) Harmonized Tripartite Guidelines (Non-Clinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals I('H M3(m), 2000 November 9), and generally accepted procedures for the testing of therapeutic agents.
TEST AND CONTROL ARTICLES
Test Article Identity and Preparation 1002531 The test article. CUR-963, is a chemically stabilized antisense oligonucleotide.
The vehicle for intravenous delivery is phosphate-buffered saline (PBS).
Vehicle characterization 4)1-[00254] For the PBS vehicle, the composition, batch number, expiry date and storage conditions (temperature and light/dark) was obtained from the supplier.
Test Article Storage and Handling [00255] The test substance and vehicle were stored according to the received storage conditions supplied by the Sponsor and manufacturer, accordingly.
Analysis of the Test Article Formulations [00256] Samples of the test article formulation will be cryopreserved for analysis of the concentration, stability and homogeneity of the test substance formulations.
TEST SYSTEM RATIONALE
[00257] The primate is a suitable non rodent species, acceptable to regulatory authorities as an indicator of potential hazards, and for which extensive background data are available. The African green monkey specifically is a highly clinically relevant model of multiple human physiologic and disease states.
[00258] The intravenous route of administration corresponds to a possible human therapeutic route. The dose of the test articles was based on the results of the dose finding studies of analogous compounds previously performed in the African green monkey.
[00259] African green monkeys were chosen as the primate of choice as the test substances' target sequences are conserved across species with 100 % homology in primates.
Additionally, the test substance is a synthetic oligonucleotide. Consequently, dosing in primates allows for a superior assessment of the efficacy of these compounds that would be more reflective of the uptake likely to be seen in humans than in any other species.
ANIMALS
Species [00260] Chlorocebus sabaeus, non-human primate Breed [00261] African green monkey indigenous to St. Kitts.
Source [00262] RxGen, Lower Bourryeau, St. Kitts, West Indies.
Expected Age [00263] The test animals were adults.
Expected Body Weight [00264] The monkeys weigh approximately 3-4 kg. The actual range may vary but will be documented in the data.
Sex .. [00265] The test animals were adult females.
Number of Animals [00266] Ten animals were screened to ensure identification of 8 animals appropriate for enrollment in the study.
Number on Study [00267] Females: 8 Justification fbr Number on Study [00268] This study was designed to use the fewest number of animals possible, consistent with the primary objective of evaluating the therapeutic efficacy of the test article in the African green monkey and prior studies of the systemic administration of this type of oligonucleotide in this species.
Animal Specification 1002691 Ten adult African Green monkeys in the weight range of 3 to 4 kg, were employed in the study. The monkeys were drug-naïve adult animals humanely trapped from the feral population that inhabits the island. Trapped monkeys were treated with antihelminthics to eliminate any possible intestinal parasite burden and were observed in quarantine for a minimum of 4 weeks prior to screening for study enrollment. The age of trapped monkeys were estimated by size and dentation, with the exclusion of older animals from the study.
Prior to study enrollment, a clinical exam was performed on each monkey, including evaluation of locomotion and dexterity. Blood samples were taken and sent to Antech Diagnostics (Memphis, TN) for comprehensive clinical chemistries and a complete blood count and lipid profiles (see sections 9.2 and 319567928 for specifications).
Monkeys with abnormal lab values, as determined by comparison to the established normal range for monkeys in the St. Kitts colony, were excluded from the study. In order to identify 8 monkeys that satisfy this criterion, 10 monkeys were screened, with the screening of additional animals as needed. Before study initiation, the selected monkeys will be transferred to individual cages to acclimate to individual housing for a one-week period. Only animals deemed suitable for experimentation will be enrolled in the study. The actual (or estimated) age and weight ranges at the start of the study will be detailed in the raw data and final report.
Animal Health and Welfare [00270] The highest standards of animal welfare were followed and adhered to guidelines stipulated by the St. Kitts Department of Agriculture and the U.S. Department of Health and Human Services. All studies will be conducted in accordance with these requirements and all applicable codes of practice for the care and housing of laboratory animals.
All applicable standards for veterinary care, operation, and review as contained in the NIH
Guide for the Care and Use of Animals. The St. Kitts facility maintains an animal research committee that reviews the protocols and inspects the facilities as required by the Guide.
The Foundation has an approved assurance filed with the Office of Laboratory Animal Welfare, as required by the Guide, #A4384-01 (Axion Research Foundation/St. Kitts Biomedical Foundation).
There are no special nonhuman primate veterinary care issues and biohazard issues raised by the research specified in this study.
Housing and Environment [00271] To allow detection of any treatment-related clinical signs, the animals were housed individually prior to surgery and postoperatively until sacrifice. The primate building in which the individual cages were situated were illuminated entirely by ambient light, which at 17 degrees north latitude approximates a 12hr:12hr light-dark cycle as recommended in the U.S.
D.H.H.S guidelines. The RxGen primate building was completely ventilated to the outside.
Additional air movement was assured by ceiling fans to maintain a constant target temperature of 23-35 C, as is typical of St. Kitts throughout the year. Twenty-four hour extremes of temperature and relative humidity (which also will not be controlled) were measured daily. During the study, the cages were cleaned at regular intervals.
Diet and Water [00272] Each animal was offered approximately 90 grams per day of a standard monkey chow diet (TekLad, Madison, WI). The specific nutritional composition of the diet was recorded. The water was periodically analyzed for microbiological purity. The criteria for acceptable levels of contaminants in stock diet and water supply were within the analytical specifications established by the diet manufacturer and the periodic facility water evaluations, respectively. The water met all criteria necessary for certification as acceptable for human consumption.
EXPERIMENTAL DESIGN
Animal Identification and Randomization [00273] Allocation was done by means of a stratified randomization procedure based on bodyweight and plasma cholesterol profiles. Prior to and after allocation to a group, each animal was identified by a tattoo on the abdomen. Tattoos are placed on all colony animals as a means of identification in the course of routine health inspections. A cage plan was drawn up to identify the individuals housed within, and individual monkeys were further identified by a labeled tag attached to their respective cage. Group sizes, doses and identification numbers [00274] The animals were assigned to 2 treatment groups, comprised of 4 monkeys in each group. Specific animal identification numbers were provided to each monkey according to the facility numbering system. This system uniquely identifies each monkey by a letter followed by a three digit number, e.g. Y032.
Route and Frequency of Administration [00275] Animals were dosed once daily on Days 1, 3, and 5 delivered intravenously by manual infusion over ¨10 min. The infusion rate will be 24 mL/kg/h. The animals were sedated with ketamine and xylazine prior to and during the dosing procedure. A
venous catheter (Terumo mini vein infusion set, 20 gauge needle, or similar appropriate infusion set) was inserted into the saphenous vein. Dosing took place in each monkey between 8:00 and 10:00 a.m. shortly after the animals wake and prior to feeding. A blood sample to assess plasma cholesterol and other lipid levels as described in Blood Chemistry section below, was collected just prior to each infusion. Blood collection preceded feeding at both sampling intervals to minimize dietary effects on cholesterol measurements.
Clinical Observations [00276] All visible signs of reaction to treatment were recorded on each day of dosing. In addition, the animals were examined at least once each week for physical attributes such as appearance and general condition.
Body Weights [00277] Body weights were recorded at weekly intervals during the treatment and post-treatment periods.
Food Consumption 1002781 Individual food consumption was not quantified. Feeding patterns were however monitored and a note made of any major changes.
Mortality and Morbidity 1002791 Mortality and morbidity will be recorded. Any decision regarding premature sacrifice will be made after consultation with the Study Director and with the Sponsor's Monitoring Scientist. if possible. Animals that are found dead or killed prematurely will be subjected to necropsy with collection of liver, kidney, heart and spleen lung tissues for histopathology. In the event of premature sacrifice a blood sample will also be taken (if possible) and the parameters determined. Animals that are found dead after regular working hours will be refrigerated overnight and necropsies performed at the start of' the next working day. If the condition of an animal requires premature sacrifice, it will be euthanized by intravenous overdose of sodium pentobarbital. All research is governed by the Principles for Use of Animals. RxGen is required by law to comply with the U.S. Department of Health and Human Services standards for primate facility, which dictates the levels of severity that the procedures within this study. specified as mild, must abide.
CLINICAL LABORATORY STUDIES
Fat Biopsies 1002801 A subcutaneous fat biopsy was performed on all study monkeys except Y775 on study days 26 by tissue extraction through a 1 cm midlinc incision inferior to the umbilicus.
TM
Biopsies were immediately immersed in a labeled cryonabe containing 2 inls of RNAlater TM
(Qiagen) and incubated at 4 C overnight, after which the RNAlater was aspirated and the sample tube flash frozen in liquid nitrogen. Following transportation in liquid nitrogen total RNA as isolated for real-time qPCR of target genes.
Rau/is:
1002811 Real time PCR results show an increase in SIRTI mRNA levels in fat biopsies from monkeys dosed with CUR-963, an oligonueleotide designed to SIRTI
antiscnse CV396200.I. compared to monkeys dosed with CUR-962 (SEQ ID NO.:53 ). alt oligonucleotide which had no effect on SIRTI expression in vitro (designed to ApoA I
antisense DA327409, data not shown). mRNA levels were determined by real time PCR
(Figure 4).
1002821 Although the invention has been illustrated and described with respect to one or more implementations. equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.
1002831 The Abstract of the disclosure will allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the following claims.
Claims (33)
1. An oligonucleotide which binds a Sirtuin 1 (SIRT1) natural antisense transcript comprising the nucleic acid sequence as set forth in any one of SEQ ID NOs: 3-6 for increasing the function or the expression of a Sirtuin 1 (SIRT1) comprising the nucleic acid sequence as set forth in SEQ ID NO: 1 or 2.
2. The oligonucleotide of claim 1, wherein the oligonucleotide comprises one of SEQ ID NOs: 7 to 48.
3. The oligonucleotide of claim 1, wherein the oligonucleotide comprises one of SEQ ID NOs: 25 to 48.
4. The oligonucleotide of claim 1, wherein the oligonucleotide comprises SEQ
ID NO: 26.
ID NO: 26.
5. The oligonucleotide of claim 1, wherein the oligonucleotide is a siRNA or is single-stranded.
6. The oligonucleotide of any one of claims 1-5, wherein the expression of Sirtuin 1 (SIRT1) is increased by at least 10% compared to a control.
7. The oligonucleotide of any one of claims 1-6, wherein the oligonucleotide further comprises at least one modification selected from the group consisting of a phosphorothioate, 2'-O-methoxyethyl (MOE), 2'-fluoro, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, carboxymethyl ester and combinations thereof.
8. The oligonucleotide of any one of claims 1-7, wherein the oligonucleotide further comprises at least one modified nucleotide selected from the group consisting of a peptide nucleic acid (PNA), a locked nucleic acid (LNA), an arabino-nucleic acid and combinations thereof.
9. The oligonucleotide of any one of claims 1-8, wherein the oligonucleotide further comprises at least one modified sugar moiety selected from the group consisting of a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety and combinations thereof.
10. The oligonucleotide of any one of claims 1-9, wherein the oligonucleotide is between 10 to 30 nucleotides in length.
11. A pharmaceutical composition comprising at least one oligonucleotide as defined in any one of claims 1-9 and a pharmaceutically acceptable excipient.
12. Use of an oligonucleotide which binds a Sirtuin 1 (SIRT1) natural antisense transcript comprising the nucleic acid sequence as set forth in any one of SEQ ID NOs: 3-6 in the manufacture of a medicament for increasing the function or expression of a Sirtuin 1 (SIRT1) comprising the nucleic acid sequence as set forth in SEQ ID NO: 1 or 2.
13. Use of an oligonucleotide which binds a Sirtuin 1 (SIRT1) natural antisense transcript comprising the nucleic acid sequence as set forth in any one of SEQ ID NOs: 3-6 for increasing the function or expression of a Sirtuin 1 (SIRT1) comprising the nucleic acid sequence as set forth in SEQ ID NO: 1 or 2.
14. The use of claim 12 or 13, wherein the oligonucleotide comprises one of SEQ ID NOs: 7 to 48.
15. The use of claim 12 or 13, wherein the oligonucleotide comprises one of SEQ ID NOs: 25 to 48.
16. The use of claim 12 or 13, wherein the oligonucleotide comprises SEQ ID
NO: 26.
NO: 26.
17. The use of claim 12 or 13, wherein the oligonucleotide is a siRNA or is single-stranded.
18. The use of any one of claims 12-17, wherein the expression of Sirtuin 1 (SIRT1) is increased by at least 10% compared to a control.
19. The use of any one of claims 12-18, wherein the oligonucleotide further comprises at least one modification selected from the group consisting of a phosphorothioate, 2'-O-methoxyethyl (MOE), 2'-fluoro, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, carboxymethyl ester and combinations thereof.
20. The use of any one of claims 12-19, wherein the oligonucleotide further comprises at least one modified nucleotide selected from the group consisting of a peptide nucleic acid (PNA), a locked nucleic acid (LNA), an arabino-nucleic acid and combinations thereof.
21. The use of any one of claims 12-20, wherein the oligonucleotide further comprises at least one modified sugar moiety selected from the group consisting of a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety and combinations thereof.
22. The use of any one of claims 12-21, wherein the oligonucleotide is between 10 to 30 nucleotides in length.
23. The use of any one of claims 12-22, wherein the oligonucleotide has at least 90% sequence identity to a complement of the Sirtuin 1 (SIRT1) natural antisense transcript.
24. A method of increasing the function or expression of a Sirtuin 1 (SIRT1) comprising the nucleic acid sequence as set forth in SEQ ID NO: 1 or 2 in patient cells or tissues in vitro comprising:
contacting said cells or tissues with an oligonucleotide which binds a Sirtuin 1 (SIRT1) natural antisense transcript comprising the nucleic acid sequence as set forth in any one of SEQ ID NOs: 3-6;
thereby increasing the expression of said SIRT1.
contacting said cells or tissues with an oligonucleotide which binds a Sirtuin 1 (SIRT1) natural antisense transcript comprising the nucleic acid sequence as set forth in any one of SEQ ID NOs: 3-6;
thereby increasing the expression of said SIRT1.
25. The method of claim 24, wherein the oligonucleotide comprises one of SEQ
ID NOs: 7 to 48.
ID NOs: 7 to 48.
26. The method of claim 24, wherein the oligonucleotide comprises one of SEQ
ID NOs: 25 to 48.
ID NOs: 25 to 48.
27. The method of claim 24, wherein the oligonucleotide comprises SEQ ID NO:
26.
26.
28. The method of claim 24, wherein the oligonucleotide is a siRNA or is single-stranded.
29. The method of any one of claims 24-28, wherein the expression of Sirtuin 1 (SIRT1) is increased by at least 10% compared to a control.
30. The method of any one of claims 24-29, wherein the oligonucleotide further comprises at least one modification selected from the group consisting of a phosphorothioate, 2'-O-methoxyethyl (MOE), 2'-fluoro, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, carboxymethyl ester and combinations thereof.
31. The method of any one of claims 24-30, wherein the oligonucleotide further comprises at least one modified nucleotide selected from the group consisting of a peptide nucleic acid (PNA), a locked nucleic acid (LNA), an arabino-nucleic acid and combinations thereof.
32. The method of any one of claims 24-31, wherein the oligonucleotide further comprises at least one modified sugar moiety selected from the group consisting of a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, a bicyclic sugar moiety and combinations thereof.
33. The method of any one of claims 24-32, wherein the oligonucleotide is between 10 to 30 nucleotides in length.
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11996508P | 2008-12-04 | 2008-12-04 | |
US61/119,965 | 2008-12-04 | ||
US15725509P | 2009-03-04 | 2009-03-04 | |
US61/157,255 | 2009-03-04 | ||
US25907209P | 2009-11-06 | 2009-11-06 | |
US61/259,072 | 2009-11-06 | ||
PCT/US2009/066445 WO2010065662A2 (en) | 2008-12-04 | 2009-12-02 | Treatment of sirtuin 1 (sirt1) related diseases by inhibition of natural antisense transcript to sirtuin 1 |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2746001A1 CA2746001A1 (en) | 2010-06-10 |
CA2746001C true CA2746001C (en) | 2020-03-31 |
Family
ID=42233852
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2746001A Active CA2746001C (en) | 2008-12-04 | 2009-12-02 | Treatment of sirtuin 1 (sirt1) related diseases by inhibition of natural antisense transcript to sirtuin 1 |
Country Status (10)
Country | Link |
---|---|
US (1) | US20110237649A1 (en) |
EP (1) | EP2370580B1 (en) |
JP (2) | JP6099868B2 (en) |
KR (1) | KR101749352B1 (en) |
CN (2) | CN108042560A (en) |
CA (1) | CA2746001C (en) |
ES (1) | ES2760912T3 (en) |
MX (1) | MX366774B (en) |
RU (1) | RU2551234C2 (en) |
WO (1) | WO2010065662A2 (en) |
Families Citing this family (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2963116B1 (en) * | 2009-03-04 | 2020-11-11 | CuRNA, Inc. | Treatment of sirtuin 1 (sirt1) related diseases by inhibition of natural antisense transcript to sirt 1 |
CA2768947C (en) * | 2009-07-24 | 2018-06-19 | Opko Curna, Llc | Treatment of sirtuin (sirt) related diseases by inhibition of natural antisense transcript to a sirtuin (sirt) |
ES2854004T3 (en) * | 2009-07-24 | 2021-09-20 | Curna Inc | Treatment of diseases related to Sirtuin 6 (SIRT6) by inhibiting the natural antisense transcript for SIRT6 |
CA2798218A1 (en) * | 2010-05-03 | 2011-11-10 | Curna, Inc. | Treatment of sirtuin (sirt) related diseases by inhibition of natural antisense transcript to a sirtuin (sirt) |
CN103025873B (en) * | 2010-06-23 | 2018-05-08 | 库尔纳公司 | SCNA relevant diseases are treated by suppressing the natural antisense transcript of voltage-gated sodium channel α subunits (SCNA) |
WO2012054723A2 (en) * | 2010-10-22 | 2012-04-26 | Opko Curna Llc | Treatment of alpha-l-iduronidase (idua) related diseases by inhibition of natural antisense transcript to idua |
US9920317B2 (en) | 2010-11-12 | 2018-03-20 | The General Hospital Corporation | Polycomb-associated non-coding RNAs |
AU2011325956B2 (en) | 2010-11-12 | 2016-07-14 | The General Hospital Corporation | Polycomb-associated non-coding RNAs |
US20150010474A1 (en) * | 2011-12-23 | 2015-01-08 | Auckland Uniservices Limited | Compounds And Methods For Selective Imaging And/Or Ablation |
CN102552912A (en) * | 2012-01-30 | 2012-07-11 | 林曙光 | Use of adrenergic beta-3-receptor retardant in tumor resistance |
US20150133527A1 (en) * | 2012-05-04 | 2015-05-14 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Regulation of cardiac sodium channels by sirt1 and sirt1 activators |
KR102028784B1 (en) | 2012-05-16 | 2019-10-04 | 트랜슬레이트 바이오 인코포레이티드 | Compositions and methods for modulating gene expression |
EA201492116A1 (en) | 2012-05-16 | 2015-05-29 | Рана Терапьютикс, Инк. | COMPOSITIONS AND METHODS FOR MODULATING THE EXPRESSION OF MECP2 |
US10837014B2 (en) | 2012-05-16 | 2020-11-17 | Translate Bio Ma, Inc. | Compositions and methods for modulating SMN gene family expression |
AU2013262700A1 (en) | 2012-05-16 | 2015-01-22 | Rana Therapeutics, Inc. | Compositions and methods for modulating hemoglobin gene family expression |
EA201492120A1 (en) | 2012-05-16 | 2015-10-30 | Рана Терапьютикс, Инк. | COMPOSITIONS AND METHODS FOR MODULATING ATP2A2 EXPRESSION |
WO2013173645A1 (en) | 2012-05-16 | 2013-11-21 | Rana Therapeutics, Inc. | Compositions and methods for modulating utrn expression |
AU2013262649A1 (en) | 2012-05-16 | 2015-01-22 | Rana Therapeutics, Inc. | Compositions and methods for modulating smn gene family expression |
TW201536329A (en) * | 2013-08-09 | 2015-10-01 | Isis Pharmaceuticals Inc | Compounds and methods for modulation of dystrophia myotonica-protein kinase (DMPK) expression |
CN103837679B (en) * | 2014-02-20 | 2016-06-08 | 浙江大学 | Sirt-1 albumen is in the application of diagnosis, prediction osteosarcoma transfer product |
WO2016070060A1 (en) | 2014-10-30 | 2016-05-06 | The General Hospital Corporation | Methods for modulating atrx-dependent gene repression |
US10900036B2 (en) | 2015-03-17 | 2021-01-26 | The General Hospital Corporation | RNA interactome of polycomb repressive complex 1 (PRC1) |
KR20170139439A (en) * | 2016-06-09 | 2017-12-19 | 단국대학교 천안캠퍼스 산학협력단 | Inhibitor of SIRT1 expression in human primary keratinocyte and the method of inhibiting SIRT1 expression using the same |
BR112019008810A2 (en) | 2016-11-01 | 2019-07-16 | Univ New York State Res Found | 5-halouracil modified microplates and their use in cancer treatment |
CN110520532B (en) * | 2017-03-31 | 2024-02-13 | 学校法人爱知医科大学 | Antisense nucleic acids that block chondroitin sulfate biosynthesis |
US11566246B2 (en) * | 2018-04-12 | 2023-01-31 | Mina Therapeutics Limited | SIRT1-saRNA compositions and methods of use |
US11312963B2 (en) * | 2018-10-18 | 2022-04-26 | Synerk Inc. | Compositions and methods for inhibiting TIGIT gene expression |
JP7450647B2 (en) * | 2019-06-26 | 2024-03-15 | エフ. ホフマン-ラ ロシュ アーゲー | Mammalian cell line with SIRT-1 gene knockout |
JP2022549611A (en) * | 2019-09-19 | 2022-11-28 | アルネー サイエンシズ、エルエルシー | Compounds and methods useful for modulating gene splicing |
Family Cites Families (177)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3687808A (en) | 1969-08-14 | 1972-08-29 | Univ Leland Stanford Junior | Synthetic polynucleotides |
US4469863A (en) | 1980-11-12 | 1984-09-04 | Ts O Paul O P | Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof |
US4534899A (en) | 1981-07-20 | 1985-08-13 | Lipid Specialties, Inc. | Synthetic phospholipid compounds |
US4426330A (en) | 1981-07-20 | 1984-01-17 | Lipid Specialties, Inc. | Synthetic phospholipid compounds |
US5023243A (en) | 1981-10-23 | 1991-06-11 | Molecular Biosystems, Inc. | Oligonucleotide therapeutic agent and method of making same |
US4476301A (en) | 1982-04-29 | 1984-10-09 | Centre National De La Recherche Scientifique | Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon |
JPS5927900A (en) | 1982-08-09 | 1984-02-14 | Wakunaga Seiyaku Kk | Oligonucleotide derivative and its preparation |
FR2540122B1 (en) | 1983-01-27 | 1985-11-29 | Centre Nat Rech Scient | NOVEL COMPOUNDS COMPRISING A SEQUENCE OF OLIGONUCLEOTIDE LINKED TO AN INTERCALATION AGENT, THEIR SYNTHESIS PROCESS AND THEIR APPLICATION |
US4605735A (en) | 1983-02-14 | 1986-08-12 | Wakunaga Seiyaku Kabushiki Kaisha | Oligonucleotide derivatives |
US4948882A (en) | 1983-02-22 | 1990-08-14 | Syngene, Inc. | Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis |
US4824941A (en) | 1983-03-10 | 1989-04-25 | Julian Gordon | Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5'-oligonucleotides in biological systems |
US4587044A (en) | 1983-09-01 | 1986-05-06 | The Johns Hopkins University | Linkage of proteins to nucleic acids |
US5118802A (en) | 1983-12-20 | 1992-06-02 | California Institute Of Technology | DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside |
US5118800A (en) | 1983-12-20 | 1992-06-02 | California Institute Of Technology | Oligonucleotides possessing a primary amino group in the terminal nucleotide |
US5550111A (en) | 1984-07-11 | 1996-08-27 | Temple University-Of The Commonwealth System Of Higher Education | Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof |
FR2567892B1 (en) | 1984-07-19 | 1989-02-17 | Centre Nat Rech Scient | NOVEL OLIGONUCLEOTIDES, THEIR PREPARATION PROCESS AND THEIR APPLICATIONS AS MEDIATORS IN DEVELOPING THE EFFECTS OF INTERFERONS |
US5258506A (en) | 1984-10-16 | 1993-11-02 | Chiron Corporation | Photolabile reagents for incorporation into oligonucleotide chains |
US5367066A (en) | 1984-10-16 | 1994-11-22 | Chiron Corporation | Oligonucleotides with selectably cleavable and/or abasic sites |
US5430136A (en) | 1984-10-16 | 1995-07-04 | Chiron Corporation | Oligonucleotides having selectably cleavable and/or abasic sites |
US4828979A (en) | 1984-11-08 | 1989-05-09 | Life Technologies, Inc. | Nucleotide analogs for nucleic acid labeling and detection |
FR2575751B1 (en) | 1985-01-08 | 1987-04-03 | Pasteur Institut | NOVEL ADENOSINE DERIVATIVE NUCLEOSIDES, THEIR PREPARATION AND THEIR BIOLOGICAL APPLICATIONS |
US5166315A (en) | 1989-12-20 | 1992-11-24 | Anti-Gene Development Group | Sequence-specific binding polymers for duplex nucleic acids |
US5405938A (en) | 1989-12-20 | 1995-04-11 | Anti-Gene Development Group | Sequence-specific binding polymers for duplex nucleic acids |
US5185444A (en) | 1985-03-15 | 1993-02-09 | Anti-Gene Deveopment Group | Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages |
US5235033A (en) | 1985-03-15 | 1993-08-10 | Anti-Gene Development Group | Alpha-morpholino ribonucleoside derivatives and polymers thereof |
US5034506A (en) | 1985-03-15 | 1991-07-23 | Anti-Gene Development Group | Uncharged morpholino-based polymers having achiral intersubunit linkages |
US4762779A (en) | 1985-06-13 | 1988-08-09 | Amgen Inc. | Compositions and methods for functionalizing nucleic acids |
US5317098A (en) | 1986-03-17 | 1994-05-31 | Hiroaki Shizuya | Non-radioisotope tagging of fragments |
JPS638396A (en) | 1986-06-30 | 1988-01-14 | Wakunaga Pharmaceut Co Ltd | Poly-labeled oligonucleotide derivative |
EP0260032B1 (en) | 1986-09-08 | 1994-01-26 | Ajinomoto Co., Inc. | Compounds for the cleavage at a specific position of RNA, oligomers employed for the formation of said compounds, and starting materials for the synthesis of said oligomers |
US5264423A (en) | 1987-03-25 | 1993-11-23 | The United States Of America As Represented By The Department Of Health And Human Services | Inhibitors for replication of retroviruses and for the expression of oncogene products |
US5276019A (en) | 1987-03-25 | 1994-01-04 | The United States Of America As Represented By The Department Of Health And Human Services | Inhibitors for replication of retroviruses and for the expression of oncogene products |
US4904582A (en) | 1987-06-11 | 1990-02-27 | Synthetic Genetics | Novel amphiphilic nucleic acid conjugates |
ATE113059T1 (en) | 1987-06-24 | 1994-11-15 | Florey Howard Inst | NUCLEOSIDE DERIVATIVES. |
US5585481A (en) | 1987-09-21 | 1996-12-17 | Gen-Probe Incorporated | Linking reagents for nucleotide probes |
US5188897A (en) | 1987-10-22 | 1993-02-23 | Temple University Of The Commonwealth System Of Higher Education | Encapsulated 2',5'-phosphorothioate oligoadenylates |
US4924624A (en) | 1987-10-22 | 1990-05-15 | Temple University-Of The Commonwealth System Of Higher Education | 2,',5'-phosphorothioate oligoadenylates and plant antiviral uses thereof |
US5525465A (en) | 1987-10-28 | 1996-06-11 | Howard Florey Institute Of Experimental Physiology And Medicine | Oligonucleotide-polyamide conjugates and methods of production and applications of the same |
DE3738460A1 (en) | 1987-11-12 | 1989-05-24 | Max Planck Gesellschaft | MODIFIED OLIGONUCLEOTIDS |
EP0348458B1 (en) | 1987-11-30 | 1997-04-09 | University Of Iowa Research Foundation | Dna molecules stabilized by modifications of the 3'-terminal phosphodiester linkage and their use as nucleic acid probes and as therapeutic agents to block the expression of specifically targeted genes |
US5403711A (en) | 1987-11-30 | 1995-04-04 | University Of Iowa Research Foundation | Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved |
US5082830A (en) | 1988-02-26 | 1992-01-21 | Enzo Biochem, Inc. | End labeled nucleotide probe |
JPH03503894A (en) | 1988-03-25 | 1991-08-29 | ユニバーシィティ オブ バージニア アランミ パテンツ ファウンデイション | Oligonucleotide N-alkylphosphoramidate |
US5278302A (en) | 1988-05-26 | 1994-01-11 | University Patents, Inc. | Polynucleotide phosphorodithioates |
US5109124A (en) | 1988-06-01 | 1992-04-28 | Biogen, Inc. | Nucleic acid probe linked to a label having a terminal cysteine |
US5216141A (en) | 1988-06-06 | 1993-06-01 | Benner Steven A | Oligonucleotide analogs containing sulfur linkages |
US5262536A (en) | 1988-09-15 | 1993-11-16 | E. I. Du Pont De Nemours And Company | Reagents for the preparation of 5'-tagged oligonucleotides |
US5512439A (en) | 1988-11-21 | 1996-04-30 | Dynal As | Oligonucleotide-linked magnetic particles and uses thereof |
US5457183A (en) | 1989-03-06 | 1995-10-10 | Board Of Regents, The University Of Texas System | Hydroxylated texaphyrins |
US5599923A (en) | 1989-03-06 | 1997-02-04 | Board Of Regents, University Of Tx | Texaphyrin metal complexes having improved functionalization |
US5354844A (en) | 1989-03-16 | 1994-10-11 | Boehringer Ingelheim International Gmbh | Protein-polycation conjugates |
US5108921A (en) | 1989-04-03 | 1992-04-28 | Purdue Research Foundation | Method for enhanced transmembrane transport of exogenous molecules |
US5391723A (en) | 1989-05-31 | 1995-02-21 | Neorx Corporation | Oligonucleotide conjugates |
US5256775A (en) | 1989-06-05 | 1993-10-26 | Gilead Sciences, Inc. | Exonuclease-resistant oligonucleotides |
US4958013A (en) | 1989-06-06 | 1990-09-18 | Northwestern University | Cholesteryl modified oligonucleotides |
US5227170A (en) | 1989-06-22 | 1993-07-13 | Vestar, Inc. | Encapsulation process |
US5451463A (en) | 1989-08-28 | 1995-09-19 | Clontech Laboratories, Inc. | Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides |
US5254469A (en) | 1989-09-12 | 1993-10-19 | Eastman Kodak Company | Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures |
US5591722A (en) | 1989-09-15 | 1997-01-07 | Southern Research Institute | 2'-deoxy-4'-thioribonucleosides and their antiviral activity |
US5527528A (en) | 1989-10-20 | 1996-06-18 | Sequus Pharmaceuticals, Inc. | Solid-tumor treatment method |
US5356633A (en) | 1989-10-20 | 1994-10-18 | Liposome Technology, Inc. | Method of treatment of inflamed tissues |
US5013556A (en) | 1989-10-20 | 1991-05-07 | Liposome Technology, Inc. | Liposomes with enhanced circulation time |
US5399676A (en) | 1989-10-23 | 1995-03-21 | Gilead Sciences | Oligonucleotides with inverted polarity |
DK0497875T3 (en) | 1989-10-24 | 2000-07-03 | Gilead Sciences Inc | 2'-modified oligonucleotides |
US5264564A (en) | 1989-10-24 | 1993-11-23 | Gilead Sciences | Oligonucleotide analogs with novel linkages |
US5264562A (en) | 1989-10-24 | 1993-11-23 | Gilead Sciences, Inc. | Oligonucleotide analogs with novel linkages |
US5292873A (en) | 1989-11-29 | 1994-03-08 | The Research Foundation Of State University Of New York | Nucleic acids labeled with naphthoquinone probe |
US5177198A (en) | 1989-11-30 | 1993-01-05 | University Of N.C. At Chapel Hill | Process for preparing oligoribonucleoside and oligodeoxyribonucleoside boranophosphates |
US5130302A (en) | 1989-12-20 | 1992-07-14 | Boron Bilogicals, Inc. | Boronated nucleoside, nucleotide and oligonucleotide compounds, compositions and methods for using same |
US5469854A (en) | 1989-12-22 | 1995-11-28 | Imarx Pharmaceutical Corp. | Methods of preparing gas-filled liposomes |
US5580575A (en) | 1989-12-22 | 1996-12-03 | Imarx Pharmaceutical Corp. | Therapeutic drug delivery systems |
US5486603A (en) | 1990-01-08 | 1996-01-23 | Gilead Sciences, Inc. | Oligonucleotide having enhanced binding affinity |
US5670633A (en) | 1990-01-11 | 1997-09-23 | Isis Pharmaceuticals, Inc. | Sugar modified oligonucleotides that detect and modulate gene expression |
US5587470A (en) | 1990-01-11 | 1996-12-24 | Isis Pharmaceuticals, Inc. | 3-deazapurines |
US5646265A (en) | 1990-01-11 | 1997-07-08 | Isis Pharmceuticals, Inc. | Process for the preparation of 2'-O-alkyl purine phosphoramidites |
US5623065A (en) | 1990-08-13 | 1997-04-22 | Isis Pharmaceuticals, Inc. | Gapped 2' modified oligonucleotides |
US5587361A (en) | 1991-10-15 | 1996-12-24 | Isis Pharmaceuticals, Inc. | Oligonucleotides having phosphorothioate linkages of high chiral purity |
US5578718A (en) | 1990-01-11 | 1996-11-26 | Isis Pharmaceuticals, Inc. | Thiol-derivatized nucleosides |
US5681941A (en) | 1990-01-11 | 1997-10-28 | Isis Pharmaceuticals, Inc. | Substituted purines and oligonucleotide cross-linking |
US5459255A (en) | 1990-01-11 | 1995-10-17 | Isis Pharmaceuticals, Inc. | N-2 substituted purines |
US5149797A (en) | 1990-02-15 | 1992-09-22 | The Worcester Foundation For Experimental Biology | Method of site-specific alteration of rna and production of encoded polypeptides |
US5220007A (en) | 1990-02-15 | 1993-06-15 | The Worcester Foundation For Experimental Biology | Method of site-specific alteration of RNA and production of encoded polypeptides |
US5214136A (en) | 1990-02-20 | 1993-05-25 | Gilead Sciences, Inc. | Anthraquinone-derivatives oligonucleotides |
WO1991013080A1 (en) | 1990-02-20 | 1991-09-05 | Gilead Sciences, Inc. | Pseudonucleosides and pseudonucleotides and their polymers |
US5321131A (en) | 1990-03-08 | 1994-06-14 | Hybridon, Inc. | Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling |
US5470967A (en) | 1990-04-10 | 1995-11-28 | The Dupont Merck Pharmaceutical Company | Oligonucleotide analogs with sulfamate linkages |
US5264618A (en) | 1990-04-19 | 1993-11-23 | Vical, Inc. | Cationic lipids for intracellular delivery of biologically active molecules |
GB9009980D0 (en) | 1990-05-03 | 1990-06-27 | Amersham Int Plc | Phosphoramidite derivatives,their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides |
DE69032425T2 (en) | 1990-05-11 | 1998-11-26 | Microprobe Corp., Bothell, Wash. | Immersion test strips for nucleic acid hybridization assays and methods for covalently immobilizing oligonucleotides |
US5610289A (en) | 1990-07-27 | 1997-03-11 | Isis Pharmaceuticals, Inc. | Backbone modified oligonucleotide analogues |
US5608046A (en) | 1990-07-27 | 1997-03-04 | Isis Pharmaceuticals, Inc. | Conjugated 4'-desmethyl nucleoside analog compounds |
US5218105A (en) | 1990-07-27 | 1993-06-08 | Isis Pharmaceuticals | Polyamine conjugated oligonucleotides |
US5541307A (en) | 1990-07-27 | 1996-07-30 | Isis Pharmaceuticals, Inc. | Backbone modified oligonucleotide analogs and solid phase synthesis thereof |
US5602240A (en) | 1990-07-27 | 1997-02-11 | Ciba Geigy Ag. | Backbone modified oligonucleotide analogs |
US5618704A (en) | 1990-07-27 | 1997-04-08 | Isis Pharmacueticals, Inc. | Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling |
US5688941A (en) | 1990-07-27 | 1997-11-18 | Isis Pharmaceuticals, Inc. | Methods of making conjugated 4' desmethyl nucleoside analog compounds |
US5677437A (en) | 1990-07-27 | 1997-10-14 | Isis Pharmaceuticals, Inc. | Heteroatomic oligonucleoside linkages |
US5138045A (en) | 1990-07-27 | 1992-08-11 | Isis Pharmaceuticals | Polyamine conjugated oligonucleotides |
US5489677A (en) | 1990-07-27 | 1996-02-06 | Isis Pharmaceuticals, Inc. | Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms |
ATE154246T1 (en) | 1990-07-27 | 1997-06-15 | Isis Pharmaceuticals Inc | NUCLEASE RESISTANT PYRIMIDINE MODIFIED OLIGONUCLEOTIDES THAT DETECTE AND MODULATE GENE EXPRESSION |
US5623070A (en) | 1990-07-27 | 1997-04-22 | Isis Pharmaceuticals, Inc. | Heteroatomic oligonucleoside linkages |
US5245022A (en) | 1990-08-03 | 1993-09-14 | Sterling Drug, Inc. | Exonuclease resistant terminally substituted oligonucleotides |
HU217036B (en) | 1990-08-03 | 1999-11-29 | Sanofi | Methods for making of compounds which can inhibit the gene expression |
US5177196A (en) | 1990-08-16 | 1993-01-05 | Microprobe Corporation | Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof |
US5512667A (en) | 1990-08-28 | 1996-04-30 | Reed; Michael W. | Trifunctional intermediates for preparing 3'-tailed oligonucleotides |
US5214134A (en) | 1990-09-12 | 1993-05-25 | Sterling Winthrop Inc. | Process of linking nucleosides with a siloxane bridge |
US5561225A (en) | 1990-09-19 | 1996-10-01 | Southern Research Institute | Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages |
CA2092002A1 (en) | 1990-09-20 | 1992-03-21 | Mark Matteucci | Modified internucleoside linkages |
US5432272A (en) | 1990-10-09 | 1995-07-11 | Benner; Steven A. | Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases |
CA2095212A1 (en) | 1990-11-08 | 1992-05-09 | Sudhir Agrawal | Incorporation of multiple reporter groups on synthetic oligonucleotides |
JP3220180B2 (en) | 1991-05-23 | 2001-10-22 | 三菱化学株式会社 | Drug-containing protein-bound liposomes |
US5539082A (en) | 1993-04-26 | 1996-07-23 | Nielsen; Peter E. | Peptide nucleic acids |
US5714331A (en) | 1991-05-24 | 1998-02-03 | Buchardt, Deceased; Ole | Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility |
US5719262A (en) | 1993-11-22 | 1998-02-17 | Buchardt, Deceased; Ole | Peptide nucleic acids having amino acid side chains |
US5371241A (en) | 1991-07-19 | 1994-12-06 | Pharmacia P-L Biochemicals Inc. | Fluorescein labelled phosphoramidites |
US5571799A (en) | 1991-08-12 | 1996-11-05 | Basco, Ltd. | (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response |
US5521291A (en) | 1991-09-30 | 1996-05-28 | Boehringer Ingelheim International, Gmbh | Conjugates for introducing nucleic acid into higher eucaryotic cells |
NZ244306A (en) | 1991-09-30 | 1995-07-26 | Boehringer Ingelheim Int | Composition for introducing nucleic acid complexes into eucaryotic cells, complex containing nucleic acid and endosomolytic agent, peptide with endosomolytic domain and nucleic acid binding domain and preparation |
EP0538194B1 (en) | 1991-10-17 | 1997-06-04 | Novartis AG | Bicyclic nucleosides, oligonucleotides, their method of preparation and intermediates therein |
US5484908A (en) | 1991-11-26 | 1996-01-16 | Gilead Sciences, Inc. | Oligonucleotides containing 5-propynyl pyrimidines |
US5359044A (en) | 1991-12-13 | 1994-10-25 | Isis Pharmaceuticals | Cyclobutyl oligonucleotide surrogates |
US5700922A (en) | 1991-12-24 | 1997-12-23 | Isis Pharmaceuticals, Inc. | PNA-DNA-PNA chimeric macromolecules |
US5565552A (en) | 1992-01-21 | 1996-10-15 | Pharmacyclics, Inc. | Method of expanded porphyrin-oligonucleotide conjugate synthesis |
US5595726A (en) | 1992-01-21 | 1997-01-21 | Pharmacyclics, Inc. | Chromophore probe for detection of nucleic acid |
FR2687679B1 (en) | 1992-02-05 | 1994-10-28 | Centre Nat Rech Scient | OLIGOTHIONUCLEOTIDES. |
US5633360A (en) | 1992-04-14 | 1997-05-27 | Gilead Sciences, Inc. | Oligonucleotide analogs capable of passive cell membrane permeation |
US5434257A (en) | 1992-06-01 | 1995-07-18 | Gilead Sciences, Inc. | Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages |
EP0577558A2 (en) | 1992-07-01 | 1994-01-05 | Ciba-Geigy Ag | Carbocyclic nucleosides having bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates |
US5272250A (en) | 1992-07-10 | 1993-12-21 | Spielvogel Bernard F | Boronated phosphoramidate compounds |
US5652355A (en) | 1992-07-23 | 1997-07-29 | Worcester Foundation For Experimental Biology | Hybrid oligonucleotide phosphorothioates |
US5583020A (en) | 1992-11-24 | 1996-12-10 | Ribozyme Pharmaceuticals, Inc. | Permeability enhancers for negatively charged polynucleotides |
US5574142A (en) | 1992-12-15 | 1996-11-12 | Microprobe Corporation | Peptide linkers for improved oligonucleotide delivery |
JP3351476B2 (en) | 1993-01-22 | 2002-11-25 | 三菱化学株式会社 | Phospholipid derivatives and liposomes containing the same |
US5476925A (en) | 1993-02-01 | 1995-12-19 | Northwestern University | Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups |
US5395619A (en) | 1993-03-03 | 1995-03-07 | Liposome Technology, Inc. | Lipid-polymer conjugates and liposomes |
GB9304618D0 (en) | 1993-03-06 | 1993-04-21 | Ciba Geigy Ag | Chemical compounds |
EP0691968B1 (en) | 1993-03-30 | 1997-07-16 | Sanofi | Acyclic nucleoside analogs and oligonucleotide sequences containing them |
WO1994022891A1 (en) | 1993-03-31 | 1994-10-13 | Sterling Winthrop Inc. | Oligonucleotides with amide linkages replacing phosphodiester linkages |
DE4311944A1 (en) | 1993-04-10 | 1994-10-13 | Degussa | Coated sodium percarbonate particles, process for their preparation and detergent, cleaning and bleaching compositions containing them |
US5462854A (en) | 1993-04-19 | 1995-10-31 | Beckman Instruments, Inc. | Inverse linkage oligonucleotides for chemical and enzymatic processes |
US5534259A (en) | 1993-07-08 | 1996-07-09 | Liposome Technology, Inc. | Polymer compound and coated particle composition |
US5417978A (en) | 1993-07-29 | 1995-05-23 | Board Of Regents, The University Of Texas System | Liposomal antisense methyl phosphonate oligonucleotides and methods for their preparation and use |
US5502177A (en) | 1993-09-17 | 1996-03-26 | Gilead Sciences, Inc. | Pyrimidine derivatives for labeled binding partners |
US5457187A (en) | 1993-12-08 | 1995-10-10 | Board Of Regents University Of Nebraska | Oligonucleotides containing 5-fluorouracil |
US5446137B1 (en) | 1993-12-09 | 1998-10-06 | Behringwerke Ag | Oligonucleotides containing 4'-substituted nucleotides |
ATE196311T1 (en) | 1993-12-09 | 2000-09-15 | Univ Jefferson | COMPOUNDS AND METHODS FOR SITE-SPECIFIC MUTATION IN EUKARYOTIC CELLS |
US5595756A (en) | 1993-12-22 | 1997-01-21 | Inex Pharmaceuticals Corporation | Liposomal compositions for enhanced retention of bioactive agents |
US5519134A (en) | 1994-01-11 | 1996-05-21 | Isis Pharmaceuticals, Inc. | Pyrrolidine-containing monomers and oligomers |
US5596091A (en) | 1994-03-18 | 1997-01-21 | The Regents Of The University Of California | Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides |
US5627053A (en) | 1994-03-29 | 1997-05-06 | Ribozyme Pharmaceuticals, Inc. | 2'deoxy-2'-alkylnucleotide containing nucleic acid |
US5625050A (en) | 1994-03-31 | 1997-04-29 | Amgen Inc. | Modified oligonucleotides and intermediates useful in nucleic acid therapeutics |
US5525711A (en) | 1994-05-18 | 1996-06-11 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Pteridine nucleotide analogs as fluorescent DNA probes |
US5543152A (en) | 1994-06-20 | 1996-08-06 | Inex Pharmaceuticals Corporation | Sphingosomes for enhanced drug delivery |
US5597696A (en) | 1994-07-18 | 1997-01-28 | Becton Dickinson And Company | Covalent cyanine dye oligonucleotide conjugates |
US5580731A (en) | 1994-08-25 | 1996-12-03 | Chiron Corporation | N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith |
US5597909A (en) | 1994-08-25 | 1997-01-28 | Chiron Corporation | Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use |
US5591721A (en) | 1994-10-25 | 1997-01-07 | Hybridon, Inc. | Method of down-regulating gene expression |
US5512295A (en) | 1994-11-10 | 1996-04-30 | The Board Of Trustees Of The Leland Stanford Junior University | Synthetic liposomes for enhanced uptake and delivery |
US5543165A (en) | 1995-06-06 | 1996-08-06 | Hill; Julie B. | Process of making a soluble tea product with champagne-like properties |
US5652356A (en) | 1995-08-17 | 1997-07-29 | Hybridon, Inc. | Inverted chimeric and hybrid oligonucleotides |
EP0817866A1 (en) * | 1996-01-26 | 1998-01-14 | Innogenetics N.V. | Method for detection of drug-induced mutations in the reverse transcriptase gene |
US6287860B1 (en) | 2000-01-20 | 2001-09-11 | Isis Pharmaceuticals, Inc. | Antisense inhibition of MEKK2 expression |
AU2003291755A1 (en) * | 2002-11-05 | 2004-06-07 | Isis Pharmaceuticals, Inc. | Oligomers comprising modified bases for binding cytosine and uracil or thymine and their use |
ATE517992T1 (en) * | 2002-11-14 | 2011-08-15 | Dharmacon Inc | FUNCTIONAL AND HYPERFUNCTIONAL SIRNA |
CL2003002353A1 (en) * | 2002-11-15 | 2005-02-04 | Vertex Pharma | COMPOUNDS DERIVED FROM DIAMINOTRIAZOLS, INHIBITORS D ELA PROTEINA QUINASA; PHARMACEUTICAL COMPOSITION; PREPARATION PROCEDURE; AND ITS USE OF THE COMPOUND IN THE TREATMENT OF DISEASES OF ALLERGIC DISORDERS, PROLIFERATION, AUTOIMMUNES, CONDIC |
US20050136395A1 (en) * | 2003-05-08 | 2005-06-23 | Affymetrix, Inc | Methods for genetic analysis of SARS virus |
US20070105109A1 (en) * | 2003-07-02 | 2007-05-10 | Geesaman Bard J | Sirt1 and genetic disorders |
EP2520652B1 (en) * | 2004-04-09 | 2015-06-10 | Genecare Research Institute Co., Ltd | Cancer cell-specific apoptosis-inducing agents that target chromosome stabilization-associates genes |
US8394947B2 (en) * | 2004-06-03 | 2013-03-12 | Isis Pharmaceuticals, Inc. | Positionally modified siRNA constructs |
US8187876B2 (en) * | 2004-07-14 | 2012-05-29 | Gamida Cell Ltd. | Expansion of stem/progenitor cells by inhibition of enzymatic reactions catalyzed by the Sir2 family of enzymes |
WO2006069592A2 (en) * | 2004-12-31 | 2006-07-06 | Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patientenzorg | Method for diagnosing an/or predicting preeclampsia and/or related disorders |
US20070213292A1 (en) * | 2005-08-10 | 2007-09-13 | The Rockefeller University | Chemically modified oligonucleotides for use in modulating micro RNA and uses thereof |
WO2007087113A2 (en) * | 2005-12-28 | 2007-08-02 | The Scripps Research Institute | Natural antisense and non-coding rna transcripts as drug targets |
US7825099B2 (en) * | 2006-01-20 | 2010-11-02 | Quark Pharmaceuticals, Inc. | Treatment or prevention of oto-pathologies by inhibition of pro-apoptotic genes |
JP2008195673A (en) * | 2007-02-14 | 2008-08-28 | Nippon Meat Packers Inc | Life prolongation-effective substance, and infection-protecting effect and vaccine effect-promoting substance, construct for assaying the above substances, and application of the above substances |
WO2009140562A1 (en) * | 2008-05-15 | 2009-11-19 | Sirtris Pharmaceuticals, Inc. | Sirt1 polymorphic variants and methods of use thereof |
US9209196B2 (en) | 2011-11-30 | 2015-12-08 | Sharp Kabushiki Kaisha | Memory circuit, method of driving the same, nonvolatile storage device using the same, and liquid crystal display device |
-
2009
- 2009-12-02 RU RU2011127211/10A patent/RU2551234C2/en not_active Application Discontinuation
- 2009-12-02 US US13/132,987 patent/US20110237649A1/en not_active Abandoned
- 2009-12-02 JP JP2011539660A patent/JP6099868B2/en active Active
- 2009-12-02 EP EP09831068.3A patent/EP2370580B1/en active Active
- 2009-12-02 CA CA2746001A patent/CA2746001C/en active Active
- 2009-12-02 ES ES09831068T patent/ES2760912T3/en active Active
- 2009-12-02 CN CN201810140950.2A patent/CN108042560A/en active Pending
- 2009-12-02 MX MX2011005914A patent/MX366774B/en active IP Right Grant
- 2009-12-02 KR KR1020117014257A patent/KR101749352B1/en active IP Right Grant
- 2009-12-02 CN CN200980156393.2A patent/CN102307997B/en active Active
- 2009-12-02 WO PCT/US2009/066445 patent/WO2010065662A2/en active Application Filing
-
2015
- 2015-08-27 JP JP2015167752A patent/JP6257563B2/en active Active
Also Published As
Publication number | Publication date |
---|---|
JP2012510815A (en) | 2012-05-17 |
CN102307997B (en) | 2018-03-30 |
MX2011005914A (en) | 2011-06-17 |
EP2370580A4 (en) | 2012-11-14 |
KR101749352B1 (en) | 2017-06-20 |
MX366774B (en) | 2019-07-24 |
RU2011127211A (en) | 2013-01-10 |
CN102307997A (en) | 2012-01-04 |
KR20110097869A (en) | 2011-08-31 |
JP2015228868A (en) | 2015-12-21 |
WO2010065662A3 (en) | 2010-09-30 |
EP2370580A2 (en) | 2011-10-05 |
ES2760912T3 (en) | 2020-05-18 |
US20110237649A1 (en) | 2011-09-29 |
WO2010065662A2 (en) | 2010-06-10 |
CA2746001A1 (en) | 2010-06-10 |
RU2551234C2 (en) | 2015-05-20 |
EP2370580B1 (en) | 2019-09-11 |
JP6257563B2 (en) | 2018-01-10 |
JP6099868B2 (en) | 2017-03-22 |
CN108042560A (en) | 2018-05-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2746001C (en) | Treatment of sirtuin 1 (sirt1) related diseases by inhibition of natural antisense transcript to sirtuin 1 | |
US9920322B2 (en) | Treatment of vascular endothelial growth factor (VEGF) related diseases by inhibition of natural antisense transcript to VEGF | |
US9611477B2 (en) | Treatment of tristetraproline (TTP) related diseases by inhibition of natural antisense transcript to TTP | |
US10519448B2 (en) | Treatment of brain derived neurotrophic factor (BDNF) related diseases by inhibition of natural antisense transcript to BDNF | |
CA2739464C (en) | Treatment of apolipoprotein-a1 related diseases by inhibition of natural antisense transcript to apolipoprotein-a1 | |
EP2424987B1 (en) | Treatment of hemoglobin (hbf/hbg) related diseases by inhibition of natural antisense transcript to hbf/hbg | |
EP2963116B1 (en) | Treatment of sirtuin 1 (sirt1) related diseases by inhibition of natural antisense transcript to sirt 1 | |
US20120295954A1 (en) | Treatment of interferon regulatory factor 8 (irf8) related diseases by inhibition of natural antisense transcript to irf8 | |
US20120295959A1 (en) | Treatment of rnase h1 related diseases by inhibition of natural antisense transcript to rnase h1 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20141117 |